Method of inducing neuronal production in the brain and spinal cord

ABSTRACT

The present invention relates to methods of inducing neuronal production in the brain, recruiting neurons to the brain, and treating a neurodegenerative condition by providing a nucleic acid construct encoding a neurotrophic factor, and injecting the nucleic acid construct intraventricularly into a subject&#39;s brain.

This application is a continuation of U.S. patent application Ser. No.09/846,588, filed May 1, 2001, which claims the benefit of U.S.Provisional Patent Application Ser. No. 60/201,230, filed May 1, 2000,which are hereby incorporated by reference in their entirety.

This invention was developed with government funding by NIH GrantsP50HL59312, RO1NS29813, and RO1NS33106. The U.S. Government may havecertain rights.

FIELD OF THE INVENTION

The present invention relates to a method of inducing neuronalproduction in the brain and spinal cord.

BACKGROUND OF THE INVENTION

Neural progenitor cells persist throughout the adult forebrainventricular zone, and have been found in species ranging from canariesto humans (Alvarez-Buylla et al., “Neuronal Stem Cells in the Brain ofAdult Vertebrates,” Stem Cells 13:263-72, (1995); Goldman, S. et al.,“Neuronal Precursor Cells of the Adult Rat Ventricular Zone Persist intoSenescence, with No Change in Spatial Extent or BDNF Response,” J.Neurobiology 32:554-566 (1997); Goldman, S. et al., “Neural Precursorsand Neuronal Production in the Adult Mammalian Forebrain,” Ann. N.Y.Acad. Sci. 835:30-55 (1997); Goldman, S. A. et al., “Strategies Utilizedby Migrating Neurons of the Postnatal Vertebrate Forebrain,” Trends inNeurosciences 21:107-114 (1998)). To the extent that neurogenesis andoligoneogenesis by these endogenous progenitors may be induced orsupported exogenously, these cells may provide a cellular substrate forrepair in the adult central nervous system (CNS). In culture,adult-derived progenitors have been found to respond to mitogens, inparticular epidermal growth factor (EGF) and fibroblast growth factor 2(FGF2), with increased division and neuronal mitogenesis (Palmer, T. D.et al, “FGF-2-Responsive Neuronal Progenitors Reside in Proliferativeand Quiescent Regions of the Adult Rodent Brain,” Mol. Cell Neurosci.6:474-86 (1995); Reynolds, B. A. et al, “Generation of Neurons andAstrocytes from Isolated Cells of the Adult Mammalian Central NervousSystem,” Science 255:1707-10 (1992); Richards, L. J. et al, “De NovoGeneration of Neuronal Cells from the Adult Mouse Brain,” Proc. Nat'l.Acad. Sci. USA 89:8591-5 (1992); Vescovi, A. L. et al, “bFGF Regulatesthe Proliferative Fate of Unipotent (neuronal) and Bipotent(neuronal/astroglial) EGF-generated CNS Progenitor Cells,” Neuron11:951-66, (1993)). Furthermore, neurons generated from them respond tobrain-derived neurotrophic factor (BDNF) with enhanced migration,maturation, and survival in vitro (Goldman, S. et al., “NeuronalPrecursor Cells of the Adult Rat Ventricular Zone Persist intoSenescence, with No Change in Spatial Extent or BDNF Response,” J.Neurobiology 32:554-566 (1997); Goldman, S. et al., “Neural Precursorsand Neuronal Production in the Adult Mammalian Forebrain,” Ann. N.Y.Acad. Sci. 835:30-55 (1997); Kirschenbaum, B. et al, “Brain-derivedNeurotrophic Factor Promotes the Survival of Neurons Arising from theAdult Rat Forebrain Subependymal Zone,” Proc. Nat'l. Acad. Sci. USA92:210-4 (1995)). Similarly, infusions of EGF and FGF2 into the adultventricular system stimulate mitotic gliogenesis and neurogenesisrespectively (Craig, C. G. et al., “In Vivo Growth Factor Expansion ofEndogenous Subependymal Neural Precursor Cell Populations in the AdultMouse Brain,” J. Neuroscience 16:2649-58 (1996); Kuhn, H. G. et al,“Epidermal Growth Factor and Fibroblast Growth Factor-2 Have DifferentEffects on Neural Progenitors in the Adult Rat Brain,” J. Neuroscience17:5820-5829 (1997)), while intraventricular infusions of BDNF canenhance neuronal migration to the olfactory bulb, rostral migratorystream and adjacent forebrain (Pencea, V. et al, “Infusion of BDNF intothe Lateral Ventricle of the Adult Rat Leads to an Increase in theNumber of Newly Generated Cells in the Fore-, Mid- and HindbrainParenchyma,” Soc. Neurosci. Abstr. 25:2045 (1999); Zigova, T. et al,“Intraventricular Administration of BDNF Increases the Number of NewlyGenerated Neurons in the Adult Olfactory Bulb,” Molec. CellularNeurosci. 11:234-245 (1998)). Although intriguing, these studies havebeen limited by the need for chronic intraventricular catheterization,with its dependence upon protein availability and stability, theuncertain tissue bioavailability of intraventricularly administeredproteins, and the risks of infection and catheter loss inherent inchronic ventriculostomy.

The striatum is the major target of the progressive neurodegenerationthat occurs in Huntington's Disease, in which the major neuron loss isthat of the striatal GABA-producing neurons. Other degenerativediseases, such as amyotrophic lateral sclerosis (ALS; also known as LouGehrig's Disease), and progressive muscular atrophy, result at least inpart from a decay of motor neurons which are located in the ventral hornof the spinal cord.

While there are some therapies available to treat the symptoms anddecrease the severity of such diseases (e.g., L-dopa to treatParkinson's Disease), there currently exists no effective treatment toprevent or reduce the degeneration of most of the above-mentionedclasses of affected neurons, or to promote their repair. Severalnaturally-occurring proteins have been identified based on their trophicactivity on various neurons. These molecules are termed “neurotrophicfactors”. Neurotrophic factors are endogenous, soluble proteins that canstimulate or regulate the production, survival, growth, and/ormorphological plasticity of neurons. (See Fallon and Laughlin,Neurotrophic Factors, Academic Press, San Diego, Calif. (1993)).

The known neurotrophic factors belong to several different proteinsuperfamilies of polypeptide growth factors based on their amino acidsequence homology and/or their three-dimensional structure (MacDonald etal., “A Structural Superfamily Of Growth Factors Containing A CystineKnot Motif,” Cell 73:421-424 (1993)). One family of neurotrophic factorsis the neurotrophin family. This family currently consists of nervegrowth factor (NGF), brain derived neurotrophic factor (BDNF),neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), and neurotrophin-6 (NT-6).

On the basis of current studies, and of their limitations in practice,it will be appreciated that a need exists for an efficient means ofdelivering neurotrophic differentiation agents to the adult ventricularzone, the site of residual progenitor cells in the adult brain.Furthermore, in view of the fact that many nervous system disorders anddiseases have no known cure, there is a need in the art for new methodsof inducing neuronal production in the adult brain, especially fortreating Huntington's Disease and other degenerative neurologicalconditions, as well as stroke and traumatic brain injury.

The present invention is directed to overcoming these and otherdeficiencies in the art.

SUMMARY OF THE INVENTION

The present invention relates to a method of inducing neuronalproduction in post-natal and adult brain and spinal cord. This involvesproviding a nucleic acid construct encoding a neurotrophic factor andinjecting the nucleic acid construct into a subject's lateral ventriclesor ventricular zone wall under conditions effective to express theneurotrophic factor and to induce neuronal production in the brain andspinal cord of subject.

The present invention also relates to a method of recruiting neurons tothe brain of a subject. This involves providing a nucleic acid constructencoding a neurotrophic factor and injecting the nucleic acid constructinto the subject's lateral ventricles or ventricular zone wall underconditions effective to express the neurotrophic factor and to recruitneurons to the brain of the subject.

The present invention also relates to a method of treating aneurodegenerative condition. This involves providing a nucleic acidconstruct encoding a neurotrophic factor and injecting the nucleic acidconstruct into a subject's lateral ventricles or ventricular zone wallunder conditions effective to treat a neurodegenerative condition.

The present invention also relates to another method of treating aneurodegenerative condition. This involves providing a neurotrophicfactor and injecting the neurotrophic factor into a subject's lateralventricles or ventricular zone wall under conditions effective to treata neurodegenerative condition.

Previous studies have reported that ependymal cells could expressadenovirally-delivered marker genes after intraventricular injection ofvirus (Bajocchi, G. et al., “Direct In Vivo Gene Transfer to EpendymalCells in the Central Nervous System Using Recombinant AdenovirusVectors,” Nature Genetics 3:229-234 (1993); Yoon, S. et al,“Adenovirus-Mediated Gene Delivery into Neuronal Precursors of the AdultMouse Brain,” Proc. Nat'l. Acad. Sci. USA 93:11974-11979 (1996), whichare hereby incorporated by reference in their entirety). However, noattempt had ever been made to utilize this strategy to delivertransgenes encoding neurotropic agents to either the ventricular zone,including its subependyma, or the endogenous precursor cells. Due to thehigh efficiency infection of, and transgene expression by, the adultependyma, intraventricular delivery of viral vectors can be used for thesustained delivery of neurotrophins not only to the ventricular zone,but also to the CSF, and hence throughout the neuraxis. The presentinvention provides a method of gene therapy that allows for widespreadproduction of BDNF by ependymal cells lining the ventricular wall whichresults in the subrogation of the ependyma into a secretory source forBDNF. This, in turn, results in the stimulation of neurogenesis in theinjected brain and an expansion of the regions into which new neuronscan be added to include areas such as the neostriatum that normallycannot replace lost neurons.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-C show the ependymal restriction of intraventricular adenoviralinfection. FIGS. 1A and 1B are sagittal sections of infected rat brain.FIG. 1A shows a single intraventricular injection of an adenoviralvector bearing a green fluorescent protein (GFP) gene, expressed underthe control of the constitutive CMV promoter, exhibiting widespreadinfection of the ventricular ependyma, bilaterally and throughout theventricular system. FIG. 1B, along the striatal and septal walls, GFPexpression is seen to be largely limited to the ventricular surface,with little subependymal and no parenchymal extension. FIG. 1C, acoronal section taken at the level of the main body of the lateralventricles, again reveals GFP expression by the infected striatal andcallosal ventricular surfaces. Unlike the striatal and septal walls, thecallosal wall shows subependymal and some parenchymal extension oflabeled cells. Abbreviations: Str, striatum; LV, lateral ventricle; CC,corpus callosum. Key: D, dorsal; V, ventral; A, anterior; P, posterior.

FIGS. 2A-D show adenoviral BDNF infection yielded high level BDNFexpression in vitro and in vivo. In FIGS. 2A and 2B, HeLa cellstransduced with AdBDNF secreted BDNF in a viral dose-dependent manner(n=3). In FIGS. 2C and 2D, AdBDNF injected animals showed sustainedexpression of high levels of BDNF in CSF, as measured on day 20 (n=5).FIGS. 2A and 2C show results in pg/ml, while FIGS. 2B and 2D are givenin pg/μg protein. Abbreviation: moi, for multiplicity of infection.

FIGS. 3A-E show AdBDNF transduced expression of BDNF and hGFP mRNA invivo. Serial sections of AdBDNF-GFP injected brain were treated withanti-sense probes for BDNF, shown in FIGS. 3A and 3D, or GFP, shown inFIGS. 3B and 3E. mRNA expression was restricted to the wall of thelateral ventricle. FIG. 3C shows a sense probe for BDNF, as control.Legend: d, dorsal; v, ventral; r, rostral; c, caudal. Scale=35 μm.

FIG. 4 is a diagram of the strategy employed to induce adult neuronalrecruitment in experimental subjects. FIG. 4A is a schematic coronalsection showing the site of injection of adenovirus into the lateralventricle. FIG. 4B shows vector E1-deleted (ΔE1) adenoviral type 5constructs used to express a dicistronic transcript of BDNF and hGFP (orhGFP alone, as a control vector) under the control of the constitutiveCMV early promoter. FIG. 4C depicts an experimental protocol whereadenovirus was injected on day 1, followed by IP injections of 100 mg/kgBrdU for the next 18 days. On day 20, cerebral spinal fluid (CSF) wasextracted for BDNF ELISA, and the brains were processed for BrdUimmunohistochemistry in tandem with phenotype-specific immunolabeling.

FIGS. 5A-E show that AdBDNF injection increased recruitment to theolfactory bulb. FIG. 5A shows the presence of BrdU+ cells in theolfactory bulbs of subject brain injected with AdBDNF:IRES:hGFP. FIG. 5Bshows the presence of BrdU+ cells in the olfactory bulbs ofAdNull:GFP-injected brain, at day 20. FIG. 5C shows a stereologicalreconstruction of BrdU+ cells. Viewed here at different mediolaterallevels of the olfactory bulb, FIG. 5C reveals substantially higher BrdU+cell densities in the olfactory subependyma and granular layers ofAdBDNF-treated rats, than in the AdGFP-injected controls, shown in FIG.5D. Arrows denote entry to rostral migratory stream. FIG. 5E shows thatthe average number of BrdU+ cells/mm³ in the olfactory bulb (n=4/group),plotted as a function of treatment, again revealing significantly highernumbers of newly generated, BrdU+ cells in AdBDNF-treated rats ascompared to control olfactory bulbs.

FIGS. 6A-C shows that AdBDNF-associated newly generated olfactory cellswere neurons. Confocal imaging confirmed that BrdU+ cells added to theolfactory bulb were almost entirely neurons, in rats injected with virus3 weeks before sacrifice, and given BrdU daily until the day before.FIG. 6A-C show merged z-dimension stacks of confocal images of BrdUco-labeling with β-III tubulin+(6A and 6B) and MAP-2+(6C) neurons. Thissuggests that the AdBDNF-associated increase in the olfactory bulb BrdUlabeling index reflected enhanced neurogenesis and/or recruitment to thebulb. Scale=25 μm.

FIG. 7 shows that the difference between AdBDNF and AdNull-treatedolfactory bulb BrdU labeling indices was significant to p<0.001. Noother comparisons based on total BrdU+ cell counts were significant.However, whereas BrdU+ cell addition to non-olfactory regions was almostentirely non-neuronal in AdNull control rats, the BrdU+ cell populationincluded newly generated neurons in several regions of theAdBDNF-injected brains. Thus, when BrdU+/β-III-tubulin+ neurons werespecifically compared between AdBDNF and AdNull-treatment groups, asignificant effect of AdBDNF on neuronal recruitment to the striatum wasnoted (see below). Abbreviations: VZ, ventricular zone; RMS, rostralmigratory stream; OB, olfactory bulb; Sep, septum; Str, neostriatum;Ctx, neocortex.

FIGS. 8A-B show AdBDNF treatment was associated with neuronal additionto the neostriatum. FIG. 8A shows sagittal (left) and coronal (right)schematics of the neostriatal region assessed for neuronal addition, inAdBDNF-injected rats and their AdNull-injected controls. FIG. 8B plotsthe mean density of β-III-tubulin+/BrdU+ cells in AdBDNF- andAdNull-injected striata, and in PBS-injected controls, at day 20 (n=4).

FIGS. 9A-L are confocal images of BrdU-labeled neurons found in theneostriata of AdBDNF-treated rats, 3 weeks after virus injection,demonstrating the induced the heterotopic addition ofBrdU+/β-III-tubulin+ neurons to the striatum. These cells wereidentified by immunostaining for both BrdU and β-III-tubulin. FIGS. 9A-Cshow a representative β-III-tubulin+/BrdU+ cell. FIG. 9A is az-dimension composite of serial 0.9 μm images, showing β-III-tubulin+and BrdU immunoreactivities. FIG. 9B, is a z-dimension series of 6×0.9μm confocal images taken 0.6 μm apart, displaying the concurrence ofBrdU and β-III-tubulin in the new neuron. FIG. 9C is a single opticalsection with reconstructed orthogonal images, as viewed from the sidesin both the xz and yz planes. In FIGS. 9D-F, another newly generated,β-III-tubulin+/BrdU+ neostriatal neuron (arrow) is seen, similarlyviewed as a z-stack composite in FIG. 9D. By way of contrast, this fieldalso includes both a non-neuronal BrdU+ cell, and a β-III-tubulin+ butBrdU-unlabeled resident neuron. Like FIGS. 9A-C, this field is alsoviewed as a series of optical sections (9E), and in orthogonalside-views (9F). FIGS. 9G-J show a pair of β-III-tubulin+/BrdU+ striatalneurons (arrows), composited in FIG. 9G with split images separatelyindicating β-III-tubulin+ and BrdU, respectively. FIGS. 9H and 91, arean optical section with orthogonal images taken at two different points,to allow individual assessment of the β-III-tubulin staining of each ofthese BrdU+ cells. Both BrdU+ nuclei are completely surrounded byβ-III-tubulin. FIG. 9J shows a series of z-dimension optical sectionsthrough these cells, again confirming the coincident expression of BrdUand β-III-tubulin. FIGS. 9K-L are low power views of the fields shown inFIGS. 9A-C and 9G-J, respectively, in order to visualize the range ofmorphologies of both resident (examples as arrowheads) and newly added(arrows) neurons. * in FIG. 9L shows a myelinated bundle passing throughthe striatal matrix. Scale=10 μm.

FIGS. 10A-I show newly recruited striatal neurons include medium spinyneurons. The BrdU+ neurons found in AdBDNF-treated striata expressedneuronal markers other than β-III-tubulin, which included NeuN. Theyalso expressed characteristic antigenic markers of medium spiny neuronsof the adult caudate-putamen, including calbindin-D28k, glutamic aciddecarboxylase (GAD67), and DARPP-32. FIGS. 10A-C show a typicalNeuN+/BrdU+ striatal neuron (arrow); local resident neurons(NeuN+/BrdU−) shown by arrowheads. FIG. 10A shows a z-dimensioncomposite of serial 1 μm images, with split (NeuN) and (BrdU) images onthe right. FIG. 10B shows a single optical section with reconstructedorthogonal images, as viewed from the sides in both the xz (top) and yz(right side) planes. FIG. 10C shows a z-dimension series, viewed as4×0.9 μm optical sections taken 0.6 μm apart. FIG. 10D shows aBrdU+/calbindin+ neuron, with calbindin indicated by the arrow. FIGS.10E-F show confocal images of a GAD67+/BrdU+ neuron in an AdBDNF-treatedstriatum. FIG. 10E shows a confocal section with reconstructedorthogonal side-views. In the orthogonal side-views, the BrdU+ nucleiremain completely surrounded by the GAD67 antigen. FIG. 10E shows az-dimension series of 4 separate 0.9 μm confocal images taken 0.6 μmapart, displayed to reveal the correspondence of BrdU and GAD67 in thesame cell at multiple z-levels. FIGS. 10G-I show analogous images of aDARPP32/BrdU+ neuron in the same striatum. FIG. 10G shows thez-dimension composite of serial 0.9 μm images, again with split imagesto show DARPP32 (arrow) and BrdU staining individually. FIG. 10H showsan optical section with reconstructed orthogonal side-views, asdescribed. In both the xz and yz planes, the BrdU+ nucleus is completelysurrounded by DARPP32 signal. FIG. 10 I shows a z-dimension seriesthrough this cell. All images were taken of striatal sections sampledfrom AdBDNF-treated rats, sacrificed 3 weeks after virus administration.Scale=10 μm.

FIG. 11 shows AdBDNF-induced striatal neurons matured and survived forat least 5-8 weeks, as demonstrated by β-III-tubulin+/BrdU+,GAD67+/BrdU+, and DARPP32+/BrdU+ striatal neurons persisted inAdBDNF-injected rats. FIGS. 11A-C show a typical β-III-tubulin+/BrdU+neuron found in an AdBDNF-treated striatum, 8 weeks after virusinjection. FIG. 11A shows the z-dimension composite of serial 1 μmimages, with split images to show β-III-tubulin+ (arrow) and BrdU,respectively. FIG. 11B shows a confocal section with reconstructedorthogonal images, as viewed from the sides in both xz (top) and yz(right side) planes. FIG. 11C shows a z-dimension series of 4 separate0.9 μm confocal images taken 0.6 μm apart, confirming theβ-III-tubulin-immunoreactivity of the BrdU+ cell. FIGS. 11D-F areanalogous images of a GAD67+/BrdU+ neuron, viewed in an AdBDNF-treatedstriatum at 8 weeks. Only one of the two adjacent GAD67+ neurons (arrow)is BrdU-labeled. FIGS. 11G-I show a representative DARPP-32+/BrdU+neuron, again found in an AdBDNF-treated striatum 8 weeks after virusinjection. FIG. 11G is a z-dimension composite of serial opticalsections, showing DARPP32 and BrdU immunoreactivities. DARPP32+/BrdU+neurons indicated by arrows; BrdU-unlabeled resident neurons indicatedby arrowheads. FIG. 11H shows orthogonal views of the DARPP-32+/BrdU+striatal neuron. FIG. 11I showing serial 0.9 μm optical sections taken0.6 μm apart, confirms the coincidence of BrdU and DARPP-32 in the samecell. Scale=10 μm.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method of inducing neuronalproduction in post-natal and adult brain and spinal cord. This involvesproviding a nucleic acid construct encoding a neurotrophic factor andinjecting the nucleic acid construct into a subject's lateral ventriclesor ventricular zone wall under conditions effective to express theneurotrophic factor and to induce neuronal production in the subject.

Neuronal production as used herein refers to the generation of newneurons. One type of nucleic acid suitable for the present invention arenucleic acids which encode growth factor products, in particularneurotrophic growth factors. Such nucleic acids include, but are notlimited to, the nucleic acid encoding BDNF, the neurotrophins NT-3(Regeneron, Tarrytown, N.Y.) and NT-4/NT-5, insulin-like growth factor,nerve growth factor (NGF), the recently identified neurotrophic familyof factors designated “NNT” (see U.S. Pat. No. 6,143,874 to Chang, whichis hereby incorporated by reference in its entirety), ciliaryneurotrophic factor (CNTF), and the interleukins.

In the brain, a protein known as bone morphogenic protein drivesprogenitor cells to differentiate into glial cells. Noggin is adevelopmental molecule which suppresses bone morphogenic protein in thebrain. Without the influence of bone morphogenic protein, progenitorcells differentiate into glia cells rather than neurons. Thus, nogginacts to induce neuronal production through its suppression of endogenousbone morphogenic protein (Lim et al., “Noggin Antagonizes BMP SignalingTo Create A Niche for Adult Neurogenesis,” Neuron 28: 713-726 (2000);Zimmerman et al., “The Spemann Organizer Signal Noggin Binds andInactivates Bone Morphogenetic Protein 4,” Cell 86: 599-606 (1996),which are hereby incorporated by reference in their entirety).Therefore, the nucleic acid which encodes the neurotrophic factor nogginis suitable for use in the nucleic acid construct of the presentinvention.

Also suitable for use in the present invention is a nucleic acid whichencodes a neurotrophic factor which is an inhibitor of bone morphogenicproteins. These factors are proteins which, like noggin, are capable ofsuppressing bone morphogenic protein, thereby driving thedifferentiation of progenitor cells in the brain into neurons. (Lim etal., “Noggin Antagonizes BMP Signaling To Create a Niche for AdultNeurogenesis,” Neuron 28: 713-726 (2000); Zimmerman et al., “The SpemannOrganizer Signal Noggin Binds and Inactivates Bone Morphogenetic Protein4,” Cell 86: 599-606 (1996), which are hereby incorporated by referencein their entirety). The suppression of bone morphogenic protein bynoggin or noggin-like proteins, as they are also known, may be usedeffectively in combination or serial addition with BDNF to furtherincrease neuronal production in the brain.

A gene or cDNA encoding the desired neurotrophic factor product orprotein, or fragment thereof, may be obtained for example by screening agenomic or cDNA library, or by PCR amplification.

Providing a nucleic acid construct of the present invention involvesincorporating the nucleic acid molecules of the present invention intohost cells using conventional recombinant DNA technology. Generally,this involves inserting the nucleic acid molecule into an expressionsystem to which the nucleic acid molecule is heterologous (i.e., notnormally present). The heterologous nucleic acid molecule is insertedinto the expression system which includes the necessary elements for thetranscription and translation of the inserted protein coding sequences.The practice of the present invention will employ, unless otherwiseindicated, conventional methods of virology, microbiology, molecularbiology, and recombinant DNA techniques within the skill of the art.Such techniques are explained fully in the literature. See, e.g.,Sambrook, et al., Molecular Cloning: A Laboratory Manual (1989); DNACloning: A Practical Approach, vol. I & II (D. Glover, ed.);Oligonucleotide Synthesis (N. Gait, ed., Current Edition); Nucleic AcidHybridization (B. Hames & S. Higgins, eds., Current Edition);Fundamental Virology, 2nd Edition, vol. I & II (B. N. Fields and D. M.Knipe, eds.), which are hereby incorporated by reference in theirentirety.

The introduction of a particular foreign or native gene into a mammalianhost is facilitated by first introducing the gene sequence into asuitable nucleic acid vector. “Vector” is used herein to mean anygenetic element, such as a plasmid, phage, transposon, cosmid,chromosome, virus, virion, etc., which is capable of replication whenassociated with the proper control elements and which is capable oftransferring gene sequences between cells. Thus, the term includescloning and expression vectors, as well as viral vectors. The nucleicacid molecules of the present invention may be inserted into any of themany available expression vectors and cell systems using reagents thatare well known in the art.

Examples of viruses which have been employed as vectors for thetransduction and expression of exogenous genes in mammalian cellsinclude the SV40 virus (Innis et al., “Chromatin Structure of SimianVirus 40-pBR322 Recombinant Plasmids in COS-1 Cells,” Mol. Cell Biol.3(12):2203-2210 (1983); Okayama et al., “Bacteriophage Lambda Vector forTransducing a cDNA Clone Library into Mammalian Cells,” Mol. Cell Biol.5(5): 1136-1142 (1985), which are hereby incorporated by reference intheir entirety) and bovine papilloma virus (Meneguzzi et al.,“Plasmidial Maintenance in Rodent Fibroblasts of a BPV1-pBR322 ShuttleVector Without Immediately Apparent Oncogenic Transformation of theRecipient Cells,” EMBO J. 3(2):365-371 (1984); DiMaio et al., “BovinePapillomavirus Vector that Propagates as a Plasmid in Both Mouse andBacterial Cells,” Proc. Nat'l. Acad. Sci. USA 79(13):4030-4034 (1982);Lusky et al., “Characterization of the Bovine Papilloma Virus PlasmidMaintenance Sequences,” Cell 36(2):391-401 (1984); Giri et al.,“Comparative Studies of the Expression of Linked Escherichia coli gptGene and BPV-1 DNAs in Transfected Cells,” Virology 127(2):385-396(1983), which are hereby incorporated by reference in their entirety),the retrovirus Moloney murine sarcoma virus (Perkins et al., “Design ofa Retrovirus-Derived Vector for Expression and Transduction of ExogenousGenes in Mammalian Cells,” Mol. Cell Biol. 3(6):1123-1132 (1983); Lee etal., “DNA Clone of Avian Fujinami Sarcoma Virus withTemperature-Sensitive Transforming Function in Mammalian Cells,” J.Virol. 44(1):401-412 (1982); Curran et al., “FBJ Murine OsteosarcomaVirus: Identification and Molecular Cloning of Biologically ActiveProviral DNA,” J. Virol. 44(2):674-682 (1982); Gazit et al., “MammalianCell Transformation by a Murine Retrovirus Vector Containing the AvianErythroblastosis Virus erbB Gene,” J. Virol. 60(1):19-28 (1986), whichare hereby incorporated by reference in their entirety), and HIV-basedviruses.

A number of adenovirus (Ad) based gene delivery systems have also beendeveloped. Human adenoviruses are double-stranded DNA viruses whichenter cells by receptor-mediated endocytosis. These viruses areparticularly well suited for gene therapy, because they are easy to growand manipulate and they exhibit a broad host range in vivo. Adenovirusis easily produced at high titers and is stable so that it can bepurified and stored. Even in the replication-competent form,adenoviruses generally cause only low level morbidity and are notassociated with human malignancies. Furthermore, Ad infects bothdividing and non-dividing cells; a number of tissues which are targetsfor gene therapy comprise largely non-dividing cells (U.S. Pat. No.6,171,855 to Askari, which is hereby incorporated by reference in itsentirety). For descriptions of various adenovirus-based gene deliverysystems, see, e.g., Haj-Ahmad et al., “Development of aHelper-Independent Human Adenovirus Vector and Its Use in the Transferof the Herpes Simplex Virus Thymidine Kinase Gene,” J. Virol.57(1):267-274 (1986); Bett et al., “Packaging Capacity and Stability ofHuman Adenovirus Type 5 Vectors,” J. Virol. 67(10):5911-5921 (1993);Mittereder et al., “Evaluation of the Efficacy and Safety of in vitro,Adenovirus-Mediated Transfer of the Human Cystic Fibrosis TransmembraneConductance Regulator cDNA,” Hum. Gene Ther. 5(6):717-729 (1994); Sethet al., “Mechanism of Enhancement of DNA Expression Consequent toCointernalization of a Replication-Deficient Adenovirus and UnmodifiedPlasmid DNA,” J. Virol. 68(2):933-940 (1994); Barr et al., “EfficientCatheter-Mediated Gene Transfer into the Heart UsingReplication-Defective Adenovirus,” Gene Ther. 1(1):51-58 (1994); Berkneret al., “Development of Adenovirus Vectors for the Expression ofHeterologous Genes,” Biotechniques 6(7):616-629 (1988); Rich et al.,“Development and Analysis of Recombinant Adenoviruses for Gene Therapyof Cystic Fibrosis,” Hum. Gene Ther. 4(4):461-476 (1993), which arehereby incorporated by reference in their entirety.

Retroviral vectors, capable of integration into the cellular chromosome,have also been used for the identification of developmentally importantgenes via insertional mutagenesis (see, e.g., U.S. Pat. No. 6,207,455 toChang, which is hereby incorporated by reference in its entirety).Retroviral vectors are also used in therapeutic applications (e.g., genetherapy), in which a gene (or genes) is added to a cell to replace amissing or defective gene or to inactivate a pathogen such as a virus.The members of the family Retroviridae are characterized by the presenceof reverse transcriptase in their virions (U.S. Pat. No. 6,207,344 toChang, which is hereby incorporated by reference in its entirety). Thefamily is divided into three subfamilies: (1) Oncovirinae, including allthe oncogenic retroviruses, and several closely related non-oncogenicviruses; (2) Lentivirinae, the “slow retroviruses,” discussed in greaterdetail below, and (3) Spumavirinae, the “foamy” retroviruses that inducepersistent infections, generally without causing any clinical disease(U.S. Pat. No. 6,218,181 to Verma et al., which is hereby incorporatedby reference in its entirety). Some of the retroviruses are oncogenic(i.e., tumorigenic), while others are not. The oncoviruses inducesarcomas, leukemias, lymphomas, and mammary carcinomas in susceptiblespecies (U.S. Pat. No. 6,033,905 to Wilson et al., which is herebyincorporated by reference in its entirety). Retroviruses infect a widevariety of species, and may be transmitted both horizontally andvertically. They are integrated into the host DNA, and are capable oftransmitting sequences of host DNA from cell to cell. This has led tothe development of retroviruses as vectors for various purposesincluding gene therapy. For example, the majority of the approved genetransfer trials in the United States rely on replication-defectiveretroviral vectors harboring a therapeutic polynucleotide sequence aspart of the retroviral genome (Miller et al., “Gene Transfer byRetrovirus Vectors Occurs Only in Cells that are Actively Replicating AtThe Time of Infection,” Mol. Cell Biol. 10(8):4239-4442 (1990); Cornettaet al., “No Retroviremia or Pathology in Long-term Follow-up of MonkeysExposed to Amphotropic Retrovirus,” Hum. Gene Ther. 2(3):215-219 (1991),which are hereby incorporated by reference in their entirety). As isknown in the art, the major advantages of retroviral vectors for genetherapy are the high efficiency of gene transfer into certain types ofreplicating cells, the precise integration of the transferred genes intocellular DNA, and the lack of further spread of the sequences after genetransfer (U.S. Pat. No. 6,033,905 to Wilson et al., which is herebyincorporated by reference in its entirety).

As used herein, the term “lentivirus” refers to a group (or genus) ofretroviruses that give rise to slowly developing disease. Virusesincluded within this group include HIV (human immunodeficiency virus;including HIV type 1, and HIV type 2), the etiologic agent of the humanacquired immunodeficiency syndrome (AIDS); visna-maedi, which causesencephalitis (visna) or pneumonia (maedi) in sheep, the caprinearthritis-encephalitis virus, which causes immune deficiency, arthritis,and encephalopathy in goats; equine infectious anemia virus, whichcauses autoimmune hemolytic anemia, and encephalopathy in horses; felineimmunodeficiency virus (FIV), which causes immune deficiency in cats;bovine immune deficiency virus (BIV), which causes lymphadenopathy,lymphocytosis, and possibly central nervous system infection in cattle;and simian immunodeficiency virus (SIV), which cause immune deficiencyand encephalopathy in sub-human primates. Diseases caused by theseviruses are characterized by a long incubation period and protractedcourse. Usually, the viruses latently infect monocytes and macrophages,from which they spread to other cells. HIV, FIV, and SIV also readilyinfect T lymphocytes (i.e., T-cells). Lentivirus virions have bar-shapednucleoids and contain genomes that are larger than other retroviruses.Lentiviruses use tRNA^(lys) as primer for negative-strand synthesis,rather than the tRNA^(pro) commonly used by other infectious mammalianretroviruses. The lentiviral genomes exhibit homology with each other,but not with other retroviruses (Davis et al., Microbiology, 4th ed.,J.B. Lippincott Co., Philadelphia, Pa., pp. 1123-1151 (1990), which ishereby incorporated by reference in its entirety). An important factorin the disease caused by these viruses is the high mutability of theviral genome, which results in the production of mutants capable ofevading the host immune response. The advantage of lentiviruses is theability for sustained transgene expression. Thus, in one embodiment ofthe present invention, a lentiviral vector is employed to providelong-term expression of the neurotrophic transgene in a target cell.

Adeno-associated viruses (AAV) may also be employed as a vector in thepresent invention. AAV is a small, single-stranded (ss) DNA virus with asimple genomic organization (4.7 kb) that makes it an ideal substratefor genetic engineering. Two open reading frames encode a series of repand cap polypeptides. Rep polypeptides (rep78, rep68, rep62, and rep40)are involved in replication, rescue, and integration of the AAV genome.The cap proteins (VP1, VP2, and VP3) form the virion capsid. Flankingthe rep and cap open reading frames at the 5′ and 3′ ends are 145 bpinverted terminal repeats (ITRs), the first 125 bp of which are capableof forming Y- or T-shaped duplex structures. Of importance for thedevelopment of AAV vectors, the entire rep and cap domains can beexcised and replaced with a therapeutic or reporter transgene (B. J.Carter, in “Handbook of Parvoviruses”, ed., P. Tijsser, CRC Press, pp.155-168 (1990), which is hereby incorporated by reference in itsentirety). It has been shown that the ITRs represent the minimalsequence required for replication, rescue, packaging, and integration ofthe AAV genome (U.S. Pat. No. 5,871,9982 to Wilson et al., which ishereby incorporated by reference in its entirety).

As noted above, viral vectors have been successfully employed in orderto increase the efficiency of introducing a recombinant vector intosuitably sensitive host cells. Therefore, viral vectors are particularlysuited for use in the present invention, including any adenoviral (Ad),retroviral, lentiviral, or adeno-associated viral (AAV) vectorsdescribed above or known in the art. Current research in the field ofviral vectors is producing improved viral vectors with high-titer andhigh-efficiency of transduction in mammalian cells (see, e.g., U.S. Pat.No. 6,218,187 to Finer et al., which is hereby incorporated by referencein its entirety). Such vectors are suitable in the present invention, asis any viral vector that comprises a combination of desirable elementsderived from one or more of the viral vectors described herein. It isnot intended that the expression vector be limited to a particular viralvector.

Certain “control elements” or “regulatory sequences” are alsoincorporated into the vector-construct. The term “control elements”refers collectively to promoter regions, polyadenylation signals,transcription termination sequences, upstream regulatory domains,origins of replication, internal ribosome entry sites (“IRES”),enhancers, and the like, which collectively provide for the replication,transcription, and translation of a coding sequence in a recipient cell.Not all of these control elements need always be present so long as theselected coding sequence is capable of being replicated, transcribed,and translated in an appropriate host cell.

The term “promoter region” is used herein in its ordinary sense to referto a nucleotide region comprising a DNA regulatory sequence, wherein theregulatory sequence is derived from a gene which is capable of bindingRNA polymerase and initiating transcription of a downstream(3′-direction) coding sequence. Transcriptional control signals ineukaryotes comprise “promoter” and “enhancer” elements. Promoter andenhancer elements have been isolated from a variety of eukaryoticsources, including genes in yeast, insect, and mammalian cells, andviruses. Analogous control elements, i.e., promoters, are also found inprokaryotes. Such elements may vary in their strength and specificity.For example, promoters may be “constitutive” or “inducible.”

A constitutive promoter is a promoter that directs expression of a genethroughout the development and life of an organism. Examples of someconstitutive promoters that are widely used for inducing expression oftransgenes include the nopoline synthase (NOS) gene promoter fromAgrobacterium tumefaciens (U.S. Pat. No. 5,034,322 to Rogers et al.,which is hereby incorporated by reference in its entirety), thecytomegalovirus (CMV) early promoter, those derived from any of theseveral actin genes, which are known to be expressed in most cells types(U.S. Pat. No. 6,002,068 to Privalle et al., which is herebyincorporated by reference in its entirety), and the ubiquitin promoter,which is a gene product known to accumulate in many cell types.

An inducible promoter is a promoter that is capable of directly orindirectly activating transcription of one or more DNA sequences orgenes in response to an inducer. In the absence of an inducer, the DNAsequences or genes will not be transcribed. The inducer can be achemical agent, such as a metabolite, or a physiological stress directlyimposed upon the organism such as cold, heat, toxins, or through theaction of a pathogen or disease agent. A recombinant cell containing aninducible promoter may be exposed to an inducer by externally applyingthe inducer to the cell or organism by exposure to the appropriateenvironmental condition or the operative pathogen.

Inducible promoters may be used in the viral vectors of this invention.These promoters will initiate transcription only in the presence of anadditional molecule. Examples of inducible promoters include thetetracycline response element and promoters derived from theβ-interferon gene, heat shock gene, metallothionein gene or anyobtainable from steroid hormone-responsive genes. Tissue specificexpression has been well characterized in the field of gene expressionand tissue specific and inducible promoters are well known in the art.These genes are used to regulate the expression of the foreign geneafter it has been introduced into the target cell.

Another type of promoter suitable for the present invention is a cellspecific promoter. “Specific,” as used herein to describe a promoter,means that the promoter permits substantial transcription of the DNAonly in a predetermined, or “chosen” cell type. A chosen cell type canrefer to different types of cells, or different stages in thedevelopmental cycle of a cell. An example of a cell specific promoteruseful in the present invention is the nestin enhancer (E/nestin). Thisderives from the 637 bp-region between bases 1162 and 1798 of the secondintronic enhancer of the rat nestin gene, which is evolutionarilyconserved between human and rat. E/nestin is sufficient to control geneexpression in CNS neuroepithelial progenitor cells (Lothian et al., “AnEvolutionarily Conserved Region in the Second Intron of the Human NestinExpression to CNS Progenitor Cells and to Early Neural Crest Cells,”Eur. J. Neurosci. 9(3):452-462 (1997), Roy et al., “Promoter TargetedSelection and Isolation of Neural Progenitor Cells from Adult HumanVentricular Zone,” J. Neurosci. Research 59: 321-331 (2000), which arehereby incorporated by reference in their entirety). In one aspect ofthe present invention, the nestin enhancer is placed upstream to a basalpromoter in order to drive gene expression specifically in neuralprecursor cells. Another example of a cell specific promoter suitablefor the present invention is the Tα1 tubulin promoter, which uses aregulatory sequence neuronal progenitor cell using a regulatory sequenceexpressed only in neuronal progenitor cells and young neurons (Roy etal., “In vitro Neurogenesis by Neural Progenitor Cells Isolated From theAdult Human Hippocampus,” Nature Medicine: 6, 271-277(2000); (Wang etal., “Isolation of Neuronal Precursors by Sorting Embryonic ForebrainTransfected Regulated by the T Alpha 1 Tubulin Promoter,” Nat.Biotechnol. 16(2):196-201 (1998), which are hereby incorporated byreference in their entirety). Also suitable in the present invention arepromoters of the musashi gene (Good et al., “The Human Musashi Homologue1 (MSI1) Gene Encoding the Homologue of musashi/Nrp-1, A NeuralRNA-Binding Protein Putatively Expressed in CNS Stem Cells AndNeuroprogenitors Cells,” Genomics 52:382-384 (1998), which is herebyincorporated by reference in its entirety), the SOX2 gene (Zapponi etal., “SOX2 Regulatory Sequences: Direct Expression of a β-geo Transgeneto Telencephalic Neural Stem Cells and Precursors of Mouse EmbryoRevealing Regionalization of Gene Expression in CNS Stem Cells,”Development 127:2368-2382 (2000), which is hereby incorporated byreference in its entirety), and the neurogenin gene (Simmons, et al.,“Neurogenin2 Expression in Ventral and Dorsal Spinal Neural TubeProgenitor Cells is Regulated by Distinct Enhancers,” DevelopmentalBiol. 229: 327-339 (2001), which is hereby incorporated by reference inits entirety), each of which is specific for neuroprogenitor cells atdifferent stages of their development.

It will be appreciated by those skilled in the art that any number ofsuitable transcriptional regulatory elements may be used to directspecific cell-type gene expression the present invention. Selection willbe highly dependent upon the vector system and host utilized.

Cell specific promoters are particularly preferable in the presentinvention, because they provide a second level of control over transgeneexpression, in addition to that of selective transduction by the vector.The most frequently used promoters are viral in origin, often derivedfrom a different virus than the vector backbone, for examplecytomegalovirus promoters have been used in all vector systems. Viralpromoters have the advantages of being smaller, stronger, and betterunderstood than most human promoter sequences.

To ensure efficient expression, 3′ polyadenylation regions must bepresent to provide for proper maturation of the mRNA transcripts. Thenative 3′-untranslated region of the gene of interest is preferablyused, but the polyadenylation signal from, for example, SV40,particularly including a splice site, which provides for more efficientexpression, could also be used. Alternatively, the 3′-untranslatedregion derived from a gene highly expressed in a particular cell typecould be fused with the gene of interest.

The vector of choice, a suitable marker gene, promoter/enhancerregion(s), and an appropriate 3′ regulatory region can be operablyligated together to produce the expression system of the presentinvention, or suitable fragments thereof, using well known molecularcloning techniques as described in Sambrook et al., Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor Press, NY (1989),and Ausubel et al. (1989) Current Protocols in Molecular Biology, JohnWiley & Sons, New York, N.Y., which are hereby incorporated by referencein their entirety. The term “operably linked” as used herein refer tothe linkage of nucleic acid sequences in such a manner that a nucleicacid molecule capable of directing the transcription of a given geneand/or the synthesis of a desired protein molecule is produced. The termalso refers to the linkage of amino acid sequences in such a manner thata functional protein is produced.

Typically, an antibiotic or other compound useful for selective growthof the transformed cells only is added as a supplement to the media. Thecompound to be used will be dictated by the selectable marker elementpresent in the plasmid with which the host cell was transformed.Suitable genes are those which confer resistance to gentamycin, G418,hygromycin, streptomycin, spectinomycin, tetracycline, chloramphenicol,and the like. Similarly, “reporter genes,” which encode enzymesproviding for production of an identifiable compound identifiable, orother markers which indicate relevant information regarding the outcomeof gene delivery, are suitable. For example, various luminescent orphosphorescent reporter genes are also appropriate, such that thepresence of the heterologous gene may be ascertained visually.

An example of a marker suitable for the present invention is the greenfluorescent protein (GFP) gene. The isolated nucleic acid moleculeencoding a green fluorescent protein can be deoxyribonucleic acid (DNA)or ribonucleic acid (RNA, including messenger RNA or mRNA), genomic orrecombinant, biologically isolated or synthetic. The DNA molecule can bea cDNA molecule, which is a DNA copy of a messenger RNA (mRNA) encodingthe GFP. In one embodiment, the GFP can be from Aequorea victoria(Prasher et al., “Primary Structure of the Aequorea VictoriaGreen-Fluorescent Protein,” Gene 111(2):229-233 (1992); U.S. Pat. No.5,491,084 to Chalfie et al., which are hereby incorporated by referencein their entirety). A plasmid encoding the GFP of Aequorea victoria isavailable from the ATCC as Accession No. 75547. Mutated forms of GFPthat emit more strongly than the native protein, as well as forms of GFPamenable to stable translation in higher vertebrates, are commerciallyavailable from Clontech Laboratories, Inc. (Palo Alto, Calif.) and canbe used for the same purpose. The plasmid designated pTα1-GFPh (ATCCAccession No. 98299) includes a humanized form of GFP. Indeed, anynucleic acid molecule encoding a fluorescent form of GFP can be used inaccordance with the subject invention. Standard techniques are then usedto place the nucleic acid molecule encoding GFP under the control of thechosen cell specific promoter.

Markers are also suitable for assessing neuronal production followinginjection. An exemplary marker for this purpose is the mitotic markerbromodeoxyuridine (BrdU). The subject can be injected with BrdU, whichis indicative of DNA replication in cells, simultaneously or followingthe injection of the nucleic acid-viral vector of the present invention.Similarly, markers specific for neurogenesis, or neuronal production,can also be assessed in spinal cord by ELISA of the subject's CSF forthe appropriate neurotrophic factor. Also suitable are markers which areindicative of the stage of development of a cell, for example, the NeuNgene, which is expressed only by mature neurons.

The selection marker employed will depend on the target species and/orhost or packaging cell lines compatible with a chosen vector.

Once the nucleic acid construct of the present invention has beenprepared and inserted into the desired vector, it is ready to beincorporated into a host cell. Basically, this method is carried out bytransforming a host cell with a nucleic construct of the presentinvention under conditions effective to yield transcription of the DNAmolecule in the host cell, using standard cloning procedures known inthe art, such as that described by Sambrook et al., Molecular Cloning: ALaboratory Manual, Second Edition, Cold Springs Laboratory, Cold SpringsHarbor, N.Y. (1989), which is hereby incorporated by reference in itsentirety. Suitable hosts include, but are not limited to, bacteria,virus, yeast, mammalian cells, insect, plant, and the like. Where thevector is a viral vector, the host cell is chosen to optimize packaging,where required, and titer. For example, where the nucleic acid of thepresent invention is inserted into an adenovirus vector, the cell lineHEK293 is an appropriate host line, with the expectation of high vectorprogeny titers. The vector DNA may be introduced into the packaging cellby any of a variety of transfection techniques, e.g., calcium phosphatecoprecipitation, electroporation, etc. (See, e.g., Sambrook, et al.,Molecular Cloning: A Laboratory Manual (1989); DNA Cloning: A PracticalApproach, vol. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N.Gait, ed., Current Edition); Nucleic Acid Hybridization (B. Hames & S.Higgins, eds., Current Edition); Fundamental Virology, 2nd Edition, vol.I & II (B. N. Fields and D. M. Knipe, eds.), which are herebyincorporated by reference in their entirety.) Other conventional methodsemployed in this invention include homologous recombination of the viralgenomes, plaquing of viruses in agar overlay, methods of measuringsignal generation, and the like known in the art or described inliterature.

Following transfection of an appropriate host with the viral vector ofthe present invention, the virus is propagated in the host andcollected. Generally, this involves collecting the cell supernatants atperiodic intervals, and purifying the viral plaques from the crudelysate, using techniques well-known in the art, for example, cesiumchloride density gradient. The titer (pfu/ml) of the virus isdetermined, and can be adjusted up (by filtration, for example), or down(by dilution with an appropriate buffer/medium), as needed. In thepresent invention, typical Ad titers are in the range of 10¹⁰-10¹²pfu/ml.

To effect the gene therapy aspect of the present invention, theisolated, purified viral vector-containing the neurotrophin-encodingnucleic acid is injected into a subject's lateral ventricles orventricular zone wall under conditions effective to express theneurotrophic factor and to induce neuronal production in the subject.“Subject” is meant herein to include any member of the class Mammaliaincluding, without limitation, humans and nonhuman primates, such aschimpanzees and other apes and monkey species; farm animals includingcattle, sheep, pigs, goats and horses; domestic animals including catsand dogs; laboratory animals including rodents such as mice, rats, andguinea pigs, and the like. The term does not denote a particular age orsex. Thus, adults and post-natal (newborn) subjects, as well as fetuses,are intended to be covered.

The recombinant viruses of the present invention may be administered toa subject, preferably suspended in a biologically compatible solution orpharmaceutically acceptable delivery vehicle. A suitable vehicleincludes sterile saline. Other aqueous and non-aqueous isotonic sterileinjection solutions and aqueous and non-aqueous sterile suspensionsknown to be pharmaceutically acceptable carriers and well known to thoseof skill in the art may be employed for this purpose.

The recombinant viruses of this invention may be administered insufficient amounts to transfect the desired cells and provide sufficientlevels of integration and expression of the selected transgene toprovide a therapeutic benefit without undue adverse effects or withmedically acceptable physiological effects which can be determined bythose skilled in the medical arts. While the preferable route ofinjection is the region of the lateral ventricle and ventricular wallzone of the subject's brain, other conventional and pharmaceuticallyacceptable parenteral routes of administration include direct deliveryto the target organ, tissue or site, intranasal, intravenous,intramuscular, subcutaneous, intradermal, and oral administration areencompassed by the present invention.

Dosages of the recombinant virus will depend primarily on factors, suchas the condition being treated, the selected gene, the age, weight, andhealth of the patient, and may thus vary among patients. Atherapeutically effective human dosage of the viruses of the presentinvention is believed to be in the range of about 5 ml of salinesolution containing concentrations of from about 2.5×10¹⁰ pfu/ml to2.5×10¹² pfu/ml virus of the present invention. Effective dosage for agiven species can be determined by correcting for differences in surfacearea of the ventricular wall. The dosage will be adjusted to balance thetherapeutic benefit against any side effects. The levels of expressionof the selected gene can be monitored to determine the selection,adjustment, or frequency of dosage administration.

The present invention also relates to a method of recruiting new neuronsto a subject's brain, including to regions of disease-related cell loss.This involves providing a nucleic acid construct encoding a neurotrophicfactor and injecting the nucleic acid construct into the subject'slateral ventricles or ventricular zone wall under conditions effectiveto express the neurotrophic factor and to recruit neurons to the brainof the subject. Preparation of the DNA construct can be carried out asdescribed above. Suitable nucleic acids include the neurotrophins givenabove, and viral propagation and injection are as described above. Thepresent invention provides a method of recruiting neurons to the brainwhich is superior to those currently existing in the art and results inthe recruitment of neurons to the olfactory bulb, the basal ganglia ofthe brain, the caudate nucleus, the putamen, and/or the globus pallidus,as well as to the to the cortex of a subject's brain.

Another aspect of the present invention relates to a method of treatinga neurodegenerative condition. This involves providing a nucleic acidconstruct encoding a neurotrophic factor and injecting the nucleic acidconstruct into a subject's lateral ventricles or ventricular zone wallunder conditions effective to treat a neurodegenerative condition,including, but not limited to, Huntington's Disease, Parkinson'sDisease, amyotrophic lateral sclerosis (ALS; also known as Lou Gehrig'sDisease), multiple sclerosis (MS), stroke, and traumatic injury to thebrain and spinal cord. Preparation of the DNA construct can be carriedout as described above. Nucleic acids suitable for this method includethe neurotrophins listed above, and viral propagation and injection arecarried out as described above.

Huntington's Disease (HD) is an autosomal dominant neurodegenerativedisease characterized by a relentlessly progressive movement disorderwith devastating psychiatric and cognitive deterioration. HD isassociated with a consistent and severe atrophy of the neostriatum whichis related to a marked loss of the GABAergic medium-sized spinyprojection neurons, the major output neurons of the striatum. Theintraventricular injections of BDNF DNA in a viral vector results in adistinct population of newly generated neurons in the neostriatum,indicating that the neurotrophic factor BDNF is particularly useful as apotential treatment for HD. Direct injection of BDNF also would beuseful in treating Huntington's Disease.

Another aspect of the present invention relates to a method of treatinga neurological condition by providing the neurotrophic factor BDNF andinjecting that factor into a subject's lateral ventricles or ventricularzone wall under conditions effective to treat the neurodegenerativecondition. In this aspect of the present invention, the BDNF may be arecombinant protein (i.e., an expression product of a BDNF-DNAconstruct). The protein in either form can be isolated by conventionalmeans, such as column purification or purification using an anti-proteinantibody.

In all aspects of the present invention, the injection of a neurotrophicfactor encoding nucleic acid into the subependyma, the cellular layerlining the ventricular cavities of the adult brain, is intended toactivate and mobilize endogenous neuroprogenitor cells of the diseasedor injured brain and spinal cord in order to restore lost brain cells.The resulting production of new neurons and the recruitment of newneurons to regions of the brain, such as the striatum and the cortex,suggest that neuronal populations may be replaceable in the brain andspinal cord of subjects suffering from neurodegenerative diseasesincluding, but not limited to, Huntington's Disease, Parkinson'sDisease, amyotrophic lateral sclerosis, and multiple sclerosis, as wellas in victims of neurological damage due to stroke or traumatic injuryto the brain and/or spinal cord.

EXAMPLES Example 1 Adenovirus Construction

An adenoviral vector was constructed bearing BDNF under the control ofthe constitutive CMV promoter, placed in tandem sequence with greenfluorescent protein (hGFP), under the control of an internal ribosomalentry site (IRES) (Morgan, R. et al, “Retroviral Vectors ContainingPutative Internal Ribosomal Entry Sites: Development of a PolycistronicGene Transfer System and Applications to Human Gene Therapy,” NucleicAcids Res. 20:1293-1299 (1992), which is hereby incorporated byreference in its entirety). In brief, BDNF cDNA (Regeneron Pharm.,Tarrytown, N.Y.) was obtained in SpeI/HindIII. A HindIII/SalI segmentcontaining an IRES site (Morgan, R. et al, “Retroviral VectorsContaining Putative Internal Ribosomal Entry Sites: Development of aPolycistronic Gene Transfer System and Applications to Human GeneTherapy,” Nucleic Acids Res. 20:1293-1299 (1992), which is herebyincorporated by reference in its entirety), and hGFP were taken frompTRUFIII (Levy, J. et al, “Retroviral Transfer and Expression of aHumanized, Red-shifted Green Fluorescent Protein Gene into Human TumorCells,” Nature Biotech. 14:610-614 (1996), which is hereby incorporatedby reference in its entirety). On digestion with SpeI-HindIII of pBDNF,the 1.1 kb TRUFIII HindIII-SalI fragment was ligated into the adenovirusshuttle vector pCMV:SV2, and digested with SpeI/SalI. The resultantconstruct was designated pAdP/CMV:BDNF:IRES:hGFP. Established methods(Graham et al., “Manipulation of Adenovirus Vectors,” In Methods inMolecular Biology, E. Murray, ed.: Humana, pp. 109-128 (1991), which ishereby incorporated by reference in its entirety) were then used toconstruct a replication-defective recombinant adenovirus, via homologousrecombination using the plasmid pJM17, which contains the E1A-deletedtype 5 adenovirus. pAd5-CMV:BDNF:IRES:hGFP was co-transfected with pJM17into HEK293 cells, and viral plaques developed for 2 weeks. Crude virallysates were then used for plaque purification. Virus was propagated inHEK293 cells, purified by cesium chloride density gradientcentrifugation, and stored at −70° C. The resultant titer of AdBDNF wasbetween 10¹¹-10¹² pfu/ml; however, both AdBDNF and its AdNull(AdCMV:hGFP) control were titered to 2.5×10¹⁰ pfu/ml before use, toensure that experimental and control rats received equivalent viralloads (see below). The efficacy of AdBDNF in driving expression of BDNFwas verified in HeLa cells by ELISA. In addition, the expression of GFPby each virus was confirmed in sections of the adult ventricular wall ofinjected rats, by direct fluorescence observation, as well as by in situhybridization of both BDNF and GFP.

Example 2 Cell Culture and In Vitro Experiments

HeLa cells were plated at a density of 1×10⁶/25 cm² in Ham's F12 with10% FBS. After 24 hours, the cells were infected with 1, 10, or 100 moi(multiplicity of infection) of AdBDNF or AdCMV:GFP, in F12 with 1% FBS.After 24 hours, serum was added to achieve a 10% concentration of FBS.After another 24 hours, the cells were washed and switched to serum-freemedia for three days. The cells and their media were then separatelycollected; the media was decanted to storage at −80° C., for later ELISAof BDNF, while the cells were collected in Ca/Mg-free HBSS/1 mM EDTA andpelleted. The cellular pellet was then resuspended in 10% FBS-containingmedia, and subjected to a viability assay; this was done to assesspotential viral toxicity in these same cultures. Triplicate 50 μlaliquots of resuspended cells were diluted in trypan blue (1:1) to labeldead cells; both live and dead cells were separately counted 20 minuteslater by hemocytometer and their ratio determined.

Example 3 Experimental Design and Stereotactic Injection

Either the Ad5CMV:BDNF:IRES:hGFP (AdBDNF) or AdCMV:hGFP (AdNull) viruseswere delivered as single 3 μl injections into the lateral ventricles of7 adult rats. Both viruses were established in the same backbone andtitered to 2.5×10¹⁰ pfu/ml before use. Using a Kopf stereotactic frame,the rats were injected at the following coordinates: AP −0.3 mm, ML ±1.2mm, DV −3.6 mm (Paxinos). The rats were injected intraperitoneally (IP)daily for 18 d thereafter with the mitotic marker bromodeoxyuridine(BrdU), 100 mg/kg. Control animals were injected with either AdCMV:GFP(AdNull; n=5) or PBS (n=5). These animals were sacrificed on the dayfollowing the last BrdU injection (day 20). Among them, 4 each of theAdBDNF, AdNull-and PBS-injected animals were used for the quantificationof BrdU+ cells in the olfactory bulb, striatum, and other regionsassessed; the remainder of the animals sacrificed on day 20 were used tosupplement our assessment of the CSF BDNF levels.

An additional sample of 3 rats was injected with AdBDNF and BrdU asnoted, but sacrificed on the 35th day after the completion of BrdUtreatment, on day 56 following viral injection. These rat brains wereexamined solely with regards to the persistence of BrdU+ neurons in theneostriatum. In all groups, daily weights were recorded beginning withthe day of viral injection, through the day of sacrifice.

Example 4 ELISA Assay of CSF and Cell Supernatants

At 20 days after viral injection, rats were injected with 0.6 ml of 65mg/ml pentobarbital, and perfused with Hank's Balanced Salt Solutionwith Mg²⁺/Ca²⁺ (Gibco-BRL, Bethesda, Md.). Cerebral spinal fluid (CSF)was collected from the cisterna magna, aliquoted, and stored at −80° C.The BDNF in the CSF of both PBS-injected and AdNull or AdBDNF-injectedrats was quantified using a two-site ELISA (Emax Immunoassay System,Promega, Madison, Wis.) (Mizisin, A. et al, “BDNF Attenuates Functionaland Structural Disorders in Nerves of Galactose-fed Rats,” J.Neuropathol. Exp. Neurol. 56:1290-1301 (1997), and Leventhal, C. et al,“Endothelial Trophic Support of Neuronal Production and Recruitment bythe Adult Mammalian Subependyma,” Molec. Cell. Neurosci. 13:450-464(1999), which are hereby incorporated by reference in their entirety).Briefly, the monoclonal anti-BDNF capture antibody did not cross reactwith other members of the neurotrophin family at concentrations up to10,000 times that used for the standard curve, while the reporterantibody was a biotinylated rabbit polyclonal anti-BDNF, similarlyselective for BDNF. The dynamic range of the ELISA was 10 pg/ml-20 ng/mlfor undiluted samples; all samples were diluted in assay buffer to bringthem into the linear range of the assay's standard curve. The CSF BDNFdeterminations were derived from 7 AdBDNF-, 5 AdNull-, and 3PBS-injected animals. Total protein levels of each CSF sample wereassessed by BCA assay (Pierce Chemical Co., Rockford, Ill.).

Cells for the supernatants of AdBDNF- and AdNull-infected HeLa celllayers, BDNF levels were reported as the average of triplicate samples.

Example 5 In Situ Hybridization

BDNF antisense and sense probes were generated from pSK-rBDNF,(Regeneron Pharmaceuticals, Tarrytown, N.Y.). The BDNF plasmid DNA waslinearized with either BamHI for the anti-sense probe or EcoRV for thesense control, then transcribed in vitro using either T7 RNA polymerasefor the anti-sense probe, or T3 RNA polymerase for the sense probe. Theantisense GFP probe was generated by linearizing pGFP with XbaI, andtranscribing in vitro with T3 RNA polymerase. The probes werenon-isotopically labeled with digoxigenin-11-UTP (BoehringerMannheim-Roche Diagnostics, Indianapolis, Ind.).

A series of 15 μm cryostat sections were permeabilized with 0.3% TritonX-100 in PBS for 15 min. The sections were dehydrated in ascendingalcohols, cleared with xylene, rehydrated, and treated with Proteinase K(1 μg/ml) for 30 min at 37° C., and postfixed with 4% paraformaldehydefor 5 min. To acetylate sections, slides were incubated for 30 min in0.1M triethanolamine (TEA) buffer, pH 8.0, containing 0.25% aceticanhydride. The sections were prehybridized with 4×SSC containing 50%formamide for 1 hr, then hybridized under coverslips for 15 h at 42° C.with digoxygenin-labeled sense or anti-sense probes (300 ng/ml) in 40%deionized formamide, 10% dextran sulfate, 1× Denhardt's, 4×SSC, 10 mMdithiothreitol, and 1 mg/ml salmon sperm DNA. After hybridization, thesections were washed in 2×SSC for 5 min to remove the coverslips, washedwith 50% formamide in 2×SSC for 20 min at 52° C., washed in 2×SSC, andtreated with RNase A (20 μg/ml) in 2×SSC for 30 min at 37° C. After 4washes in 2×SSC, the sections were washed with 0.2×SSC at 55° C. for 1hr.

For detection of digoxygenin-labeled probes the slides were washed inTris buffered-saline (TBS: 0.1M Tris HCl with 150 mM NaCl), 3×5 min,blocked in 0.1% Triton X-100 and 2% sheep serum for 30 min, andincubated overnight at 4° C. in AP-conjugated anti-digoxygenin (1:100,Boehringer Mannheim). After washing with TBS, the sections were switchedto detection buffer (100 mM Tris-HCl, pH 9.5, with 100 mM NaCl, 50 mMMgCl2) for 10 min, and incubated in NBT-BCIP solution (Bio-Rad Labs,Hercules, Calif.) with 1 mM levamisole for 2-20 hrs in the dark. Uponcolor development, the reaction was terminated by washing (3×5 min) inTBS with 10 mM EDTA.

Example 6 Immunohistochemistry

The animals were sacrificed, perfusion fixed and their brains removed oneither the 20th or 56th day after viral injection. Fixation wasaccomplished using 4% paraformaldehyde in 0.1M phosphate buffer (PB; pH7.4), with a 90 min post-fix followed by immersion and sinking in 30%sucrose in PB. All brains were cut as 15 μm sagittal sections thatincluded the olfactory bulb and rostral migratory stream rostrally.These sections were stained for BrdU, using immunoperoxidase detectionwhen staining for BrdU alone, or double-immunofluorescence when stainingfor both BrdU and neuronal markers. Individual sections were denaturedin 2N HCl for an hour, then stained for BrdU, using rat anti-BrdUantibody at 1:200 (Harlan Bioproducts, Indianapolis, Ind.), followedserially by fluorescein-conjugated anti-rat IgG at 1:150 (Jackson Labs,Bar Harbor, Me.). The sections were then washed and stained for eitherβ-III-tubulin, using the TuJ1 monoclonal antibody (Lee, M. et al.,“Posttranslational Modification of Class III b-tubulin,” Proc. Nat'l.Acad. Sci. USA 87:7195-7199 (1990), which is hereby incorporated byreference in its entirety); MAP-2, using rabbit anti-MAP2 (Bernhardt, R.et al., “Light and Electron Microscopic Studies of the Distribution ofMicrotubule-Associated Protein 2 in Rat Brain: A Difference BetweenDendritic and Axonal Cytoskeletons,” J. Comp. Neurol. 226:203-21 (1984),which is hereby incorporated by reference in its entirety); NeuN(Chemicon, Temecula, Calif.) (Eriksson, P. et al., “Neurogenesis in theAdult Human Hippocampus,” Nature Medicine 4:1313-1317 (1998), which ishereby incorporated by reference in its entirety); GAD67 (Sigma, St.Louis, Mo.); calbindin-D28K (Guan, J et al., “Selective NeuroprotectiveEffects with IGF-1 in Phenotypic Striatal Neurons Following IschemicBrain Injury in Fetal Sheep,” Neuroscience 95:831-839 (1999), which ishereby incorporated by reference in its entirety) (Sigma, St. Louis,Mo.); or DARPP-32 (Ivkovic, S. et al, “Expression of the StriatalDARPP-32/ARPP-21 Phenotype in GABAergic Neurons Requires Neurotrophinsin Vivo and In Vitro,” J. Neurosci. 19:5409-5419 (1999), which is herebyincorporated by reference in its entirety), each as previously described(Eriksson, P. et al., “Neurogenesis in the Adult Human Hippocampus,”Nature Medicine 4:1313-1317 (1998); Goldman, S. A. et al., “In VitroNeurogenesis by Neuronal Precursor Cells Derived from the Adult SongbirdBrain,” J. Neuroscience 12:2532-41 (1992); Guan, J et al., “SelectiveNeuroprotective Effects with IGF-1 in Phenotypic Striatal NeuronsFollowing Ischemic Brain Injury in Fetal Sheep,” Neuroscience 95:831-839(1999); Ivkovic, S. et al, “Expression of the Striatal DARPP-32/ARPP-21Phenotype in GABAergic Neurons Requires Neurotrophins in Vivo and InVitro,” J. Neurosci. 19:5409-5419 (1999); Menezes, J. R. et al,“Expression of Neuron-specific Tubulin Defines a Novel Population in theProliferative Layers of the Developing Telencephalon,” J Neurosci.14:5399-416 (1994); Roy, N. et al, “In Vitro Neurogenesis by NeuralProgenitor Cells Isolated from the Adult Human Hippocampus,” NatureMedicine 6:271-277 (2000), which are hereby incorporated by reference intheir entirety). All anti-mouse secondary antibodies were pre-absorbedagainst rat IgG to avoid nonspecific staining.

Example 7 Confocal Imaging

In sections double-stained for BrdU together for either β-III-tubulin,MAP-2, NeuN, DARPP-32, GAD67 or calbindin-D28, single BrdU+ cells thatappeared to be double-labeled for both the neuronal antigen and BrdUwere further evaluated by confocal imaging. Using a Zeiss LSM510confocal microscope, images were acquired in both red and green emissionchannels using an argon-krypton laser. The images were then viewed asstacked z-dimension images, both as series of single 0.9 μm opticalsections, and as merged images thereof. The z-dimension reconstructionswere all observed in profile, as every BrdU+ cell double-labeled with aneuronal marker was then observed orthogonally in both the vertical andhorizontal planes. Only after 3 observers independently deemedindividual cells as double-labeled, with central BrdU immunoreactivitysurrounded by neuronal immunoreactivity at all observation angles, inevery serial optical section, and in each merged and rotated composite,were the cells scored as double-labeled, newly-generated neurons.

Example 8 Scoring and Quantification

Unbiased counting was used to score the number, density, anddistribution of BrdU+ cells in the injected brains, using an opticaldissector procedure (Kuhn, H. G. et al, “Epidermal Growth Factor andFibroblast Growth Factor-2 Have Different Effects on Neural Progenitorsin the Adult Rat Brain,” J. Neuroscience 17:5820-5829 (1997); Sterio,D., “The Unbiased Estimation of Number and Sizes of Arbitrary ParticlesUsing the Dissector,” J. Microsc. 134:127-136 (1984); West, M.,“Stereological Methods for Estimating the Total Number of Neurons andSynapses: Issues of Precision and Bias,” Trends in Neurosci. 22:51-61(1998), which are hereby incorporated by reference in their entirety).To estimate the number of BrdU-labeled cells per region, 22 15 μmsections/animal were sampled, for both experimental and control brains;for each, every sixth section was analyzed, at 90 μm intervals. Thefirst section of each sagittal series was chosen randomly, from a totalsample that was accumulated beginning with the first appearance of theolfactory cortex on cresyl violet stained alternate sections. Typically,the sampled region included that subtended by the stereotacticcoordinates L0.3-2.3 bilaterally. By this means, a 2 mm mediolateralsegment in the sagittal plane, centered on the RMS, was sampled.

The absolute number of total BrdU+ cells in every eighth 15 μm sagittalsection was counted in each of 6 regions: 1) the anterior surface of theventricular zone (VZ), 2) the olfactory subependyma of the rostralmigratory stream (RMS), 3) the olfactory bulb, 4) the medial septum, 5)the neostriatum, and 6) the frontal cortex overlying the corpuscallosum, rostral to the perpendicular extension of the rostral-mostwall of the lateral ventricle.

In each sampled section, every BrdU+ nucleus was counted in each scoredregion; the positions of each of these cells were entered manually intoBioQuant image analysis software with its incorporated topographyreconstruction package, and the results tabulated. For each region, theresults were reported as the mean number of BrdU+ cells/section. Inaddition, for the olfactory bulb and the neostriatum, these counts wereconverted into BrdU+ cells/mm³ after determining the surface areas andhence volumes of each scored region (Michel, R. et al, “Application ofthe Cavalieri Principle and Vertical Sections Method to Lung: Estimationof Volume and Pleural Surface Area,” J. Microsc. 150:117-136 (1988),which is hereby incorporated by reference in its entirety). Statisticalanalysis was then accomplished by analysis of variance (ANOVA) followedby post-hoc Boneferroni t-tests.

To estimate total striatal neuronal number, the number of striatalneurons were counted in each of 6 age and sex-matched rats, 3 of whichwere treated with AdBDNF and the other 3 with AdNull as a negativecontrol (n=3). From each, eight 15 μm sections were analyzed,representing every 32nd sequentially, thereby sampling at 480 μmintervals beginning with the first appearance of rostrocaudally-orientedstriatal fascicles on cresyl violet stained alternate sections. In thesecresyl violet-stained sections, the number of neurons in each striatumwere counted under high magnification using morphological criteria forneuronal identity, by an observer blind with regard to the experimentalgroup. To this end, neurons were defined as large cells of >10 μmdiameter, with pale central nuclei and central nucleoli, in an otherwisebasophilic cytoplasm. To ensure the validity of these criteria, 2sections from each set of 8 were destained by acidified ethanol, thenimmunostained for calbindin. The number of calbindin-defined striatalneurons was then counted and compared to that obtained in the samesection by cresyl violet. A >98% concordance was found in the neuronalcounts obtained using these 2 methods.

Example 9 Intraventricular Delivery of Adenoviral CMV:hGFP RestrictedTransgene Expression to the Adult Ependyma and Subependyma

To first assess the distribution of adenoviral transduction following asingle intraventricular injection of virus, adenovirus bearing the geneencoding green fluorescent protein (hGFP), placed under the control ofthe CMV promoter, was injected into the lateral ventricles of 4 adultSprague-Dawley rats. The rats were sacrificed either 1 or 3 weeks later,and their brains prepared for histology. As shown in FIG. 1, it wasfound that in all 4 rats, most ependymal and scattered subependymalcells labeled heavily to single viral injection, with virtually theentire lining of the lateral ventricle noted to express GFP afterinjection of 3×10⁷ pfu adenovirus (3 μl of 10¹⁰ pfu/ml). Littleparenchymal expression of GFP was noted, despite the lack of specificityof the CMV promoter, suggesting that viral penetration outside of thesubependyma was minimal. The restriction of transgene expression to theventricular wall suggested that ependymal cells might be targetedselectively on spatial grounds alone, even without benefit ofcell-specific promoters.

Example 10 AdBDNF-Infected Cells Expressed BDNF In Vitro

The effects of adenovirally-delivered BDNF on the adult ventricular zonepool were assessed as follows. A 6 μl type 5 adenovirus was thusconstructed to express BDNF, under the control of the constitutivelyactivated CMV promoter; the virus also included hGFP as a reporter,placed under IRES promoter control. The resultant vector,AdCMV:BDNF:IRES: hGFP (hereafter referred to as AdBDNF) wascharacterized first by infecting HeLa cells, which typically do notexpress BDNF. The production of BDNF by the infected HeLa cells wasassessed as a function of time after infection, using ELISA of BDNFsecreted into the culture media. BDNF release in response toAdBDNF-infection was compared to that of both untransfected andAdCMV:hGFP (AdNull)-infected control cells, as shown in FIGS. 2A-B.Within 2 days after infection with 10 pfu/cell AdBDNF, 234±54.5 ng/ml ofBDNF protein were measured in the culture supernatant, roughly 250-foldthe levels observed in the uninfected (0.8±1.0 ng/ml) andAdNull-infected (1.0±0.6 ng/ml) control cultures. Thus, AdBDNF directedhigh-level expression of BDNF by HeLa cells.

To ensure that transgene expression was not accomplished at the expenseof cell viability, trypan blue inclusion was assessed as a function ofviral dose over the range 1-10 moi/cell. Results of the trypan blueexclusion tests showed that adenovirus-associated toxicity was minimal,and statistically insignificant over this dose range. In addition, toensure that this dose range was no more toxic for primary brain cellsthan for HeLa cells, the effect of increasing viral dose on theviability of primary adult human astrocytes, obtained from temporallobes resected from adult epileptic patients (see Leventhal, C. et al,“Endothelial Trophic Support of Neuronal Production and Recruitment bythe Adult Mammalian Subependyma,” Molec. Cell. Neurosci 13:450-464(1999), which is hereby incorporated by reference in its entirety) wasalso determined. It was found that astrocytes exposed to 10 moi AdBDNFexhibited no significant increment in lethal toxicity at 48 hrs afterinfection.

Example 11 CSF Levels of BDNF Rose Markedly Following IntraventricularInjection of AdBDNF

To assess the level of release of BDNF protein into the CSF ofAdBDNF-treated rats, a total of 14 animals were injected with eitherAdBDNF (n=7), AdNull (n=5), or PBS (n=2); all were subjected to cisternamagna puncture for CSF withdrawal at 3 weeks after viral infection. Inthe AdBDNF-injected animals, ELISAs revealed that CSF BDNF levelsaveraged 1.07±0.3 μg/g protein (mean±SE), when assessed 3 weeks afterinjection, as shown in FIGS. 2C-D. This represented 2.02±0.6 ng BDNF/mlof ventricular CSF, a level at the lower end of the dose range (2-40ng/ml) appropriate for eliciting trkB-mediated biological effects invitro (Lindsay, R. M. et al, “Neurotrophic Factors: From Molecule toMan,” Trends in Neurosciences 17:182-90 (1994), which is herebyincorporated by reference in its entirety). In contrast, BDNF wasundetectable in both the PBS and AdCMV:GFP controls (p=0.025 by ANOVA[F=5.24; 2, 13 d.f.]). The absence of detectable BDNF in theAdNull-injected controls indicated that the BDNF levels achieved in theCSF of AdBDNF-treated animals was a product of the virally-encoded BDNFtransgene. Thus, adenoviral transduction of the adult ventricularependyma permitted high-level delivery of BDNF to the brain and CSF,with expression that was sustained for at least 3 weeks after viralinfection.

Example 12 Adenoviral-Transduced BDNF mRNA Restricted to VentricularWall

In situ hybridization, using RNA probes for BDNF and hGFP, revealed thatAdCMV:BDNF:IRES:GFP transduced expression of both BDNF and hGFP mRNA invivo. Strikingly, BDNF and GFP mRNAs were largely restricted to the wallof the lateral ventricular system, as shown in FIG. 3. Even whenassessed 3 weeks after viral injection, cells overexpressing BDNF andGFP were largely limited to the ventricular wall, with little or noinfiltration of the rostral migratory stream or bulb. Thus, at leastrostrally along the anterior face of the ventricle, the infected cellpool appeared to be ependymal, with little or no direct infection ofsubependymal neuronal migrants. This pattern appeared to be maintainedalong most of the rostrocaudal extent of the ventricular system,throughout which virally-transduced BDNF and GFP mRNAs were limited tothe ependymal surface, except at the rostral tip of the lateralventricles, where scattered subependymal labeling was also noted.

Example 13 AdBDNF Infection of the Ventricular Wall IncreasedSubstantially the Number of New Neurons In Both the Rostral MigratoryStream and Olfactory Bulb

To follow the generation and fates of new neurons generated from theAdBDNF-treated ventricular zone, an initial cohort of rats were injectedwith either AdBDNF or AdNull (n=4/group). These injections were followedwith daily intraperitoneal injections of BrdU, at 100 mg/kg for the next18 days. On day 20, the animals were sacrificed, CSF was extracted forBDNF ELISA, and the brains fixed along with the olfactory bulbs. Thebrains were then sectioned and stained for BrdU in tandem withphenotype-specific markers. This strategy is detailed in FIGS. 4A-C.

The effects of AdBDNF on neuronal recruitment were first assessed in theregion of the rostral migratory stream, as measured posteriorly from thestriatum and its ventricular wall up to, and including, the internalgranular layer of the olfactory bulb. Within this region, the incidenceof BrdU+ cells rose from 3398±346 cells/mm³ (±SE) in the control animalsto 8288±1199 cells/mm3 in the AdBDNF-treated rats. FIGS. 5A and 5B showthe presence of BrdU+ cells in AdBDNF-treated brains and control brains,respectively. Within the olfactory bulb itself (measured rostrally fromthe line connecting the dorsal and ventral posterior borders of theolfactory cortex), AdBDNF treatment increased by 2.44±0.1-fold thenumber of BrdU+ cells, relative to the AdNull controls (p=0.0006 byANOVA [F=42.1; 1, 7 d.f.]), as seen in FIGS. 5C-D. This value reflectedthe cell counts obtained from scoring entire sagittal sections of theolfactory bulb. The values included both the internal and externalgranular zones of the scored bulbs. As shown in FIG. 5E, the number ofcells migrating to the olfactory bulb was substantially greater in theAdBDNF-treated animals than their AdNull controls.

In both the AdBDNF and AdNull animals, the BrdU+ cells weredouble-immunostained for β-III-tubulin and/or MAP-2, to establish theproportion of neurons within the total BrdU+ cell pool. In both groups,BrdU-incorporating cells found within the olfactory stream almostinvariably expressed β-III-tubulin-immunoreactivity, as shown in FIG. 6.The same was true in the olfactory bulb, within which double-labeledcells for MAP-2/BrdU were also frequent. Interestingly, MAP-2-labeledBrdU+ cells were seen only in the olfactory bulb, and not in theolfactory subependyma or migratory stream. Instead, these cells werefirst noted within the granular layer of the olfactory bulb itself,consistent with the differentiation of mitotic β-III-tubulin+neuroblasts to postmitotic MAP-2+ neurons upon terminal migration fromthe olfactory subependyma to the olfactory cortex (Goldman, S. A. etal., “Strategies Utilized by Migrating Neurons of the PostnatalVertebrate Forebrain,” Trends in Neurosciences 21:107-114 (1998); Lois,C. et al, “Chain Migration of Neuronal Precursors,” Science 271:978-981(1996), which are hereby incorporated by reference in their entirety).Quantitatively, in the AdNull-treated rats, 93.2±0.5% of BrdU+ cells inthe olfactory bulb expressed β-III-tubulin. This proportion wasvirtually identical to that obtained in the AdBDNF-treated olfactorybulbs, in which an average of 93.0±1.8 and 89.4±2.4% of BrdU+ cellsco-expressed neuronal β-III-tubulin or MAP-2, respectively. These dataindicate that most cells recruited to the olfactory bulbs were neurons,and that AdBDNF substantially promoted the addition of these new neuronsto the adult olfactory system.

Example 14 Confocal Imaging Confirmed Cells Added to the Olfactory Bulbswere Neurons

High-magnification confocal imaging confirmed the neuronal antigenicityof the BrdU-labeled cells in both the rostral migratory stream andolfactory bulb. Representative sections were taken from 4 brains,including 2 AdBDNF-treated experimental animals and 2 AdNull controlanimals. Mid-sagittal sections derived from each of these weredouble-immunostained for BrdU together with either β-III-tubulin orMAP-2, and imaged via confocal laser scanning, with compositing andreconstruction in the z-dimension to ensure the neuronalimmunoreactivity of BrdU+ cells. This confirmed that those BrdU+ cellsadded to the olfactory bulb were almost entirely neurons, in that theyexpressed MAP-2 as well as β-III-tubulin, and did so in both the AdBDNFand AdNull-treated animals. As shown in FIG. 6, merged z-stacks ofconfocal images of MAP-2+ and B-III tubulin+ neurons, co-labeled forBrdU, confirmed that >90% all BrdU+ nuclei in the olfactory bulb wereharbored by MAP-2+ or β-III tubulin+ cells. This indicated that theAdBDNF-associated increases in the olfactory bulb BrdU labeling indicesreflected enhanced neurogenesis and/or recruitment in the treatedanimals.

Example 15 Ventricular AdBDNF Infection Induced Striatal NeuronalRecruitment

Despite the extraordinary increase in olfactory neuronal recruitment inAdBDNF-treated rats, this treatment was not associated withsignificantly increased cell division outside of the olfactory system.As shown in FIG. 7, the mean numbers of BrdU+ cells per section in thefrontal cortex, septum, and striatum were all roughly equivalent in theAdBDNF- and AdNull-injected brains, when assessed 20 days after viralinjection. Nonetheless, this left open the possibility that AdBDNF mightbe influencing either the lineage choice of mitotically-activeprogenitors, or the selective survival of their neuronal daughters. Toassess this possibility, the incidence of β-III-tubulin+/BrdU+ cells ineach non-olfactory region studied were scored, in both AdBDNF and AdNulltreated brains. When BrdU+ cells were identified by epifluorescencemicroscopy, they were subjected to two color confocal imaging withserial sections in the z plane, to estimate the incidence ofdouble-labeled β-III-tubulin+/BrdU+ cells, while ensuring that the BrdU+nuclei indeed belonged to β-III-tubulin+ cell profiles.

Only very rare BrdU+/β-III-tubulin+ neurons were found in the frontalcortex of AdBDNF-treated animals, too few to merit systematic comparisonto null controls. No examples of newly generated septal neurons werefound in either the AdBDNF or AdNull-injected animals. Surprisingly theAdBDNF treated animals were found to harbor a distinct population ofnewly generated neurons in the neostriatum, as shown in FIG. 8 and FIG.9. These BrdU+ neurons comprised a distinct minority of the BrdU+striatal cells in these brains. They were scattered throughout thestriatum, though they were most often located in its periventricularthird. Confocal imaging confirmed examples of newly generated, BrdU+striatal cells that expressed a variety of independent markers ofneuronal phenotype, that included β-III tubulin, NeuN, GAD67, DARRP-32,and calbindin-D28K. FIG. 9 shows the AdBDNF induced heterotopic additionof BrdU+/β-III-tubulin+ neurons to the striatum. FIG. 10 shows theexpression of neuronal markers of NeuN, GAD67, DARRP-32, andcalbindin-D28K in the newly recruited striatal neurons. FIG. 11 showsthat expression markers of BrdU+/β-III-tubulin+, BrdU+/GAD67, andBrdU+/DARRP-32 persist 5-8 weeks after AdBDNF injection.

Quantitatively, unbiased counting of all striatal BrdU+ cells insagittal sections of AdBDNF-treated animals revealed an average of1663±748 BrdU+ cells/mm³. Among a randomly chosen sample of 477 BrdU+striatal cells located in sections (n=17) selected from 3 AdBDNF-treatedbrains, 41 cells (8.3±2.3%) could be confirmed as double-labeled forboth BrdU and β-III-tubulin by confocal imaging, as shown in FIG. 9.This compared to the complete absence of double-labeled striatal neuronsin the PBS-injected rats (0/95 cells, n=8 sections taken from 3 rats),and to the relatively rare incidence of BrdU+/β-III-tubulin+ neuronsobserved in the AdNull-injected rats—15 among 591 randomly chosen BrdU+striatal cells (2.1±1.1%; n=23 sections taken from 6 rats, shown in FIG.8B. Analysis of variance (ANOVA) established that the incidence of newstriatal neurons in the AdBDNF-injected rats and their controls differedsignificantly (p=0.006; F=9.48 [2, 9 d.f.]). On a per animal basis,8.3±2.3% of the BrdU+ cells in the AdBDNF-treated rat striata, or143±26.5 cells/mm³, were antigenically-definable as neurons, as shown inFIG. 8B. This represented only 0.34% (=143/41,637) of the total striatalneuronal pool. However, since these cells were generated in just 3weeks, one might predict that proportionately more neurons may be addedto the striatum with longer survival times (see below), provided thatboth viral BDNF expression and progenitor cell competence are sustained.

Example 16 No Neurogenesis Noted in Untreated Adult Striatum

To assess the incidence, if any, of neuronal addition to the normaluntreated neostriatum, control animals that were injectedintraventricularly with either PBS (n=3) or AdNull (n=5), who receivedBrdU on days 2 through 19, and which were then sacrificed on the nextday (3 wk time point) were examined. In the PBS-injected rats, noBrdU+/β-III-tubulin+ striatal neurons were found, as shown in FIG. 8B.In these same rats, constitutive neurogenesis was observed in both theolfactory bulb and dentate gyrus, as would be expected, though noattempt was made at quantifying baseline neuronal recruitment at thesesites. Thus, no evidence was found of constitutive neurogenesis in thenormal unstimulated neostriatum, in contrast to the robust neuronalrecruitment observed in the AdBDNF-treated striatum.

Example 17 Adenoviral Infection per se Associated with a Minor Inductionof Neuronal Recruitment

Interestingly, and in contrast to the absence of striatal neuronaladdition noted in the PBS-treated rats, the AdNull-injected controls didexhibit a small amount of constitutive neuronal addition to thestriatum. As noted above, among 591 BrdU+ striatal cells identified inAdNull-injected rats sacrificed at 3 weeks, confocal analysis revealedthat 15 cells (2.1±1.1%) double-labeled for BrdU and β-III-tubulin. Thiswas determined using the same criteria as in the concurrent analysis ofAdBDNF-treated striata, and the cells were assessed by the sameindividuals, who were blinded as to treatment group. As noted above, andshown in FIG. 8B, this incidence of striatal neuronal addition in theAdNull-treated rats was significantly less (p=0.006) than the 8.3±2.2%noted in their AdBDNF-treated counterparts. Nonetheless, the verypresence of BrdU+/β-III-tubulin+ neurons in the AdNull-treated striatawas surprising, given the absence of any new striatal neurons in thePBS-injected rats, and suggested that adenoviral infection itself mighthave resulted in some mobilization of neural progenitors. This raisedthe possibility that virally-induced ependymal cytokines may influencesubependymal neuronal production or migration; this in turn might allowotherwise heterotopic neuronal recruitment. Nonetheless, the substantialincrease in striatal neuronal addition in the AdBDNF-treated rats,relative to their AdNull-treated controls, suggested that anyadenovirus-associated mobilization of neural progenitor cells was minorrelative to that specifically attributable to BDNF. Together, theseobservations indicated that AdBDNF induced the addition of new neuronsto the neostriatum, an otherwise atypical site for neuronal recruitmentin the adult brain.

Example 18 AdBDNF-Induced Striatal Neurons Expressed Antigens of MediumSpiny Neurons

To assess the neuronal phenotype induced by AdBDNF infection, sectionsof AdBDNF-treated brains were immunostained for a number of markers ofstriatal phenotype. As shown in FIG. 10, some BrdU+ striatal cellsexpressed calbindin-D28K, a marker of medium spiny neurons of thecaudate-putamen (Burke, R. et al., “Relative Loss of the StriatalStriosome Compartment, Defined by Calbindin-D28 Immunostaining,Following Developmental Hypoxic-Ischemic Injury,” Neuroscience56:305-315 (1993); Waldvogel, H. et al, “Differential Sensitivity ofCalbindin and Parvalbumin Immunoreactive Cells in the Striatum toExcitotoxins,” Brain Res. 546:329-335 (1991), which are herebyincorporated by reference in their entirety). Similarly, also shown inFIG. 10, abundance of BrdU+ striatal cells that co-expressed glutamicacid decarboxylase (GAD67), a characteristic marker for GABAergicneurons, were identified.

Despite their expression by medium spiny neurons, both calbindin andGAD67 are expressed by other cell types, and even within the striatummay not be definitive markers of medium spiny neurons. Thus, to betterascertain the phenotype of AdBDNF-induced striatal neurons,double-staining for DARPP-32 (dopamine and cAMP-regulatedphosphoprotein, of 32 kDa), a highly selective marker of medium spinyneurons (Ivkovic, S. et al, “Expression of the Striatal DARPP-32/ARPP-21Phenotype in GABAergic Neurons Requires Neurotrophins in Vivo and InVitro,” J. Neurosci 19:5409-5419 (1999), which is hereby incorporated byreference in its entirety), was carried out on sections derived from thesame animals that expressed calbindin and GAD67. Among the ratssacrificed 3 weeks after virus injection, 6 of a random sample of 125BrdU+ striatal cells (4.8%) were found to be DARPP-32+, and wereconfirmed as such by confocal imaging and serial reconstruction, asshown in FIG. 10. This compared to 33 of 377 BrdU+ cells (8.8%) inadjacent sections of the same rats that expressed β-III-tubulin.Importantly, the percentage of DARPP-32+ cells among the BrdU+ striatalcell population increased with time, such that when assessed in ratssacrificed 8 weeks after AdBDNF injection, 10 of 128 BrdU+ cells (7.8%)were DARPP-32+(see below). Together, these observations suggested thatmany, if not most, of the AdBDNF-induced striatal cells matured to aphenotype characteristic of medium spiny neurons. These data raise thepossibility that AdBDNF treatment might contribute to the restoration ofthis phenotype, a critical mediator of striatopallidal communication,whose significance is underscored by its selective loss in Huntington'sDisease.

Example 19 AdBDNF-Induced Striatal Neurons Matured and Survived

The 3 week time point used to establish neuronal recruitment in responseto AdBDNF allowed the possibility that those cells generated anddetected at 3 weeks were merely transitional phenotypes, perhapstransient in their very existence. To establish the more prolongedsurvival of AdBDNF-associated striatal neurons, a distinct group ofanimals that were sacrificed and assessed 8 weeks after viral injection.Both β-III-tubulin+/BrdU+ and DARPP32+/BrdU+ double-labeled striatalcells persisted in these rats, with little apparent loss, as shown inFIG. 11. Among a sample of 106 BrdU+ cells in 3 8-week rat striatarandomly sampled for confocal imaging, 7 cells (6.6%) were found toexpress β-III-tubulin. Similarly, 10 of 128 sampled BrdU+ cells (7.8%)expressed DARPP-32. The rough equivalency of the proportion of BrdU+striatal cells that were β-III-tubulin+ and DARPP+ argued that by 8weeks after AdBDNF injection, or 5 weeks after the last BrdUincorporation, virtually all of the BrdU+ neurons, as defined byβ-III-tubulin, also would have been expected to express DARPP-32. Thus,a substantial number of AdBDNF-induced striatal neurons survived,depending upon the time point of their generation during the BrdUinjection course, for at least 5-8 weeks after terminal mitosis.Furthermore, these surviving neurons matured sufficiently to expressDARPP-32, a relatively mature marker of striatal neuronal phenotype. Assuch, these AdBDNF-induced neurons did not appear to constitutetransitional phenotypes.

Example 20 AdBDNF Treatment Associated with Systemic Weight Loss

The AdBDNF-injected animals were noted to experience a stereotypicweight loss during the 3 week period between AdBDNF delivery andsacrifice. This was not unexpected, since weight loss has previouslybeen described in rats receiving intraventricular BDNF infusions. Thissyndrome appears to be central in origin, and reflects BDNF-associatedappetite suppression and hypophagia, rather than any hypermetabolicstate (Pelleymounter, M. et al, “Characteristics of BDNF-induced WeightLoss,” Exp. Neurol. 131:229-238 (1995), which is hereby incorporated byreference in its entirety). It was found that this was not an effect ofthe virus, in that neither AdNull nor PBS-injected animals experiencedsimilar weight loss. Whereas AdNull-injected controls rose from 300±16 gat the time of viral injection, to 339±6 g at the time of sacrifice 3weeks later (n=4), a matched set of AdBDNF-treated animals lost weightduring that period, falling from 328±34 to 288±17 g/animal (p=0.016 byANOVA, comparing the slopes of weight gain as a function of time betweenAdBDNF, AdNull and PBS-treated rats [F=6.17; 2, 13 d.f.])

The time course of weight loss in the AdBDNF-treated animals suggestedthat virally-delivered BDNF was exerting rapid and powerful biologicaleffects on the target nervous system, within the same time frame as theBDNF-associated rise in neuronal recruitment. Whether this anorexicphenotype was a consequence of olfactory neuronal addition and alteredolfactory perception, or was instead an unrelated effect of central BDNFoverexpression, remains to be established.

Infection of the adult rat ventricular lining with an adenoviral BDNFexpression vector is shown herein to induce the recruitment of newneurons from resident progenitor cells of the forebrain ventricularzone. In particular, adenoviral infection resulted in the diffusetransduction of the adult ventricular wall, with the effectivesubrogation of the ependyma into a source of secreted BDNF to both theCSF and periventricular parenchyma. This resulted in the sustained,high-level secretion of BDNF by the ventricular wall, and was associatedwith a >2.4-fold increase in the recruitment of new neurons to the ratolfactory bulb over the 3 weeks following viral administration.Importantly, AdBDNF administration was also associated with theheterotopic addition of new neurons to the neostriatum, with therecruitment of BrdU-incorporatingβ-III-tubulin+/DARPP-32+/NeuN+/GAD67+/calbindin-D28K+ neurons to thestriata of AdBDNF-treated animals. These experiments comprise the firstuse of viral gene delivery as a means to induce neurogenesis fromresident progenitor cells in the adult CNS. In addition, they presentthe first evidence for induced neuronal addition to the matureneostriatum. Thus, the present invention indicates that viraltransduction of the adult ependyma to overexpress BDNF may be aneffective means of inducing the recruitment of new neurons to permissiveregions of the mature brain.

Predominantly ependymal cell expression of both BDNF and GFP mRNAs wereobserved after intraventricular injection of AdBDNF:IRES:GFP.Nonetheless, occasional subependymal labeling was noted, particularlyalong the subcallosal and dorsolateral walls of the lateral ventricles.When present, subependymal GFP fluorescence appeared as rapidly asependymal cell labeling; both were evident by 7 days after virusinjection, as shown in FIG. 1. GFP+ cells were limited to theventricular layers, though, and were never noted in either the olfactorysubependyma or striatal parenchyma, except for migrants into the corpuscallosum. Despite this restriction of virally-expressed BDNF to theventricular wall, newly generated neurons, derived from uninfectedsubependymal cells, were profoundly influenced in their production andsurvival by their genesis adjacent to AdBDNF-infected ependymal cells.Thus, the effects of ependymal BDNF on neurogenesis and neuronalrecruitment to the olfactory bulb likely derived from a paracrine effectof ependymal BDNF upon uninfected subependymal progenitors. Furthermore,BDNF's effects were presumably exerted early in the ontogeny of thesecells, before their departure from the ventricular wall, since it isunclear whether ependymal secretion of BDNF to the CSF andperiventricular parenchyma would have influenced BDNF in the olfactorybulb itself. Indeed, the mature olfactory bulb harbors high levels ofBDNF, while the neurotrophin appears relatively sequestered from theadult ventricular zone and olfactory subependyma. Thus, a likelyscenario is that ependymal BDNF acts to promote the earlydifferentiation and survival during migration of newly generated,subependymally-derived neurons, and that these cells survive to migrateinto an already BDNF-rich environment in the olfactory bulb. As such,neuronal mitogenesis and departure from the ventricular wall may beviewed as the initial rate-limiting steps for neuronal recruitment, withthe cells finding a permissive environment for survival once in thebulb.

Importantly, AdBDNF injection was also associated with the addition ofnew neurons to the neostriata of treated animals. Such neuronal additionto non-granule cell populations has only rarely been reported in theadult mammalian brain, specifically in the visual cortex (Kaplan, M.,“Proliferation of SubepenDymal Cells in the Adult Primate CNS:Differential Uptake of Thymidine by DNA-Labeled Precursors,” J.Hirnforsch 23:23-33 (1983), which is hereby incorporated by reference inits entirety) and macaque frontal cortex (Gould, E. et al.,“Neurogenesis in the Neocortex of Adult Primates,” Science 286:548-552(1999), which is hereby incorporated by reference in its entirety), aswell as in response to injury in the adult mouse frontal cortex (Magavi,S. et al, “Induction of Neurogenesis in the Neocortex of Adult Mice,”Nature 405:951-955 (2000), which is hereby incorporated by reference inits entirety). More generally though, reports of neurogenesis in theadult mammalian brain have been limited to olfactory, hippocampal, andcerebellar granule cell populations (reviewed in Goldman, S. A. et al.,“Strategies Utilized by Migrating Neurons of the Postnatal VertebrateForebrain,” Trends in Neurosciences 21:107-114 (1998), which is herebyincorporated by reference in its entirety). Nonetheless, carefulanalysis of serially-reconstructed confocal images revealed thatAdBDNF-injected animals harbored a discrete cohort of antigenicallyconfirmed neurons that co-labeled with BrdU and were scatteredthroughout the neostriatum. Since the adult neostriatum typically doesnot add new neurons, the induced neuronal addition associated withAdBDNF treatment may be viewed as heterotopic in nature.

Even though their numbers were small relative to the much larger pool ofAdBDNF-induced olfactory neurons, the recruitment kinetics of theinduced striatal pool were surprisingly robust. Given an averagestriatal neuronal BrdU labeling index of 0.34%, and an average of1.03×10⁶±6.56×10⁴ neurons per striatum, roughly 3.5×10³ neurons may beadded to each striatum over an 18 day period of BrdU injection, orapproximately 195 neurons/striatum/day. This estimate is crude andlikely an underestimate in that it is predicated on the assumptions thatdaily BrdU injections label the entire mitotic pool and that no striatalcells are dying during this period. In addition, these numbers mayreflect but one point on the dose-response curve relating neuronalrecruitment to BDNF expression levels. It is important to remember thatin the above examples neither the dose of adenovirus, nor that of itsexpressed BDNF were perturbed. Nonetheless, these numbers suggest thatAdBDNF-induced neuronal addition may be sufficiently robust tocontribute meaningfully to striatal function, if not architecture.Furthermore, the identification of many AdBDNF-induced neurons asDARPP-32+/GAD67+/calbindin-D28K+ suggests that at least a significantfraction of these neurons may be homologous to the medium spinyinterneuron population of the adult neostriatum. Since this is theneuronal population lost in Huntington's Disease and the striatonigraldegenerations, transduction of the ventricular wall with BDNF expressionvectors might be envisaged as a feasible strategy for restoringdiminished neuronal populations in the striatal degenerations, as wellas in other conditions of acquired striatal neuronal loss.

Interestingly, it was noted that the AdNull (AdCMV:hGFP)-injectedcontrols exhibited a small amount of constitutive neuronal addition tothe striatum. This did not appear to reflect neurogenesis in the normalstriatum, since PBS-injected rats exhibited no striatal neuronaladdition whatsoever. Rather, these results suggested that adenoviralinfection per se might have been sufficient to instigate mobilization ofneural progenitors. Although minor in extent and significantly lessrobust than AdBDNF-induced neuronal recruitment, the AdNull induction ofstriatal neuronal addition may represent a hitherto unrecognized featureof central viral infection, especially of the ependyma/subependyma.Presumably, virally-induced ependymal cytokines might stimulatesubependymal neurogenesis, and thereby permit otherwise heterotopicneuronal recruitment. This possibility is strengthened by reports thatadenovirally-induced cytokines include IL-6 and IL-8, both of which havebeen found to be neurotrophic in vitro (Driesse, M. et al, “Intra-CSFAdministered Recombinant Adenovirus Causes an Immune Response-MediatedToxicity,” Gene Therapy 7:1401-1409 (2000), which is hereby incorporatedby reference in its entirety). Indeed, such paracrine activation ofneurotrophic cytokines might explain recent observations of bothinflammation and apoptosis-related neuronal recruitment in the adultbrain (Magavi, S. et al, “Induction of Neurogenesis in the Neocortex ofAdult Mice,” Nature 405:951-955 (2000); Wang, Y. et al, “CorticalInterneurons Upregulate Neurotrophins In Vivo in Response to TargetedApoptotic Degeneration of Neighboring Pyramidal Neurons,” Exp. Neurol.154:389-402 (1998), which are hereby incorporated by reference in theirentirety). In any event, the significant increase in neuronalrecruitment to the striatum in the AdBDNF-treated rats, relative totheir AdNull-treated controls, argued that any virus-associated cellgenesis paled beside that specifically associated with BDNF.

It is worth noting that despite the frequent observation ofBrdU-incorporating cells in the septa, striata, and frontal cortices ofthese animals, no significant differences were noted between the AdBDNFand control animals in their BrdU-labeled cell numbers, as shown in FIG.7. As shown in FIGS. 8 and 9, AdBDNF treatment was associated with anincrease in the relative proportion of neurons among the BrdU+ cells ofthe neostriatum. Nonetheless, the percentage of confocal-validated newneurons in the overall striatal BrdU+ cell population was so small—just8% of the BrdU+ population—that AdBDNF would not have been expected toyield readily demonstrable treatment-related differences in either thetotal BrdU+ cell number or overall striatal neuronal number. Theinduction of striatal neurogenesis by AdBDNF might therefore reflecteither the neuronal differentiation of postmitotic daughters that mightotherwise have become glia, or the postmitotic rescue of daughtersotherwise destined to die. Indeed, although a number of studies havefailed to observe any mitogenic effect of BDNF on ventricular zoneprogenitor cells (Ahmed et al.,. “BDNF Enhances the Differentiation butnot the Survival of CNS Stem Cell-Derived Neuronal Precursors,” JNeurosci. 15:5765-78 (1995); Kirschenbaum, B. et al, “Brain-derivedNeurotrophic Factor Promotes the Survival of Neurons Arising from theAdult Rat Forebrain Subependymal Zone,” Proc Nat'l Acad Sci USA 92:210-4(1995), which are hereby incorporated by reference in their entirety),these data do not allow one to rule out a direct mitogenic effect invivo.

It is important to also consider the possibility that BDNF might act notonly to recruit a ventricular zone-derived population, but also toactivate resident parenchymal glial progenitors to differentiate asneurons. Studies of both adult rat (Palmer, T. et al, “FGF2 Activates aLatent Neurogenic Program in Neural Stem Cells from Diverse Regions ofthe Adult CNS,” J. Neurosci. 19:8487-8497 (1999), which is herebyincorporated by reference in its entirety) and human (Roy, N. et al,“Identification, Isolation and Enrichment of Oligodendrocyte ProgenitorCells from the Adult Human Subcortical White Matter,” J. Neurosci.19:9986-9995 (1999), which is hereby incorporated by reference in itsentirety) brain have indicated the ability of white matter progenitorcells to differentiate as neurons in vitro. In the AdBDNF-treatedneostriata in particular, the possibility of AdBDNF-induced neurogenesisfrom parenchymal progenitors is suggested by the frequent observation ofclustered pairs of BrdU+ neurons (e.g., FIG. 9G), although continueddivision of ventricular zone migrants might also explain thisobservation (Menezes, J. R. et al, “The Division of Neuronal ProgenitorCells During Migration in the Neonatal Mammalian Forebrain,” Mol CellNeurosci 6:496-508 (1995), which is hereby incorporated by reference inits entirety). Thus, while the possibility that AdBDNF might stimulateneuronal recruitment from parenchymal progenitors is intriguing, theabove data do not yet allow the source or migration routes ofAdBDNF-induced striatal neurons to be addressed.

The implications of AdBDNF-induced striatal neurogenesis may beprofound, particularly for disorders such as Huntington's Disease andstriatonigral degeneration, in which the loss of striatal neurons maydictate the pathology. The apparent assumption of a medium spinyneuronal phenotype by many, and perhaps most, AdBDNF-induced neostriatalneurons is especially intriguing, in that it suggests the potentialtherapeutic utility of this neuronal population. Axiomatically, if thesecells prove functional and able to survive, then AdBDNF-induced striatalneurons might be able to delay, abrogate, or reverse striatalneurodegenerative disease. Nonetheless, it remains to be seen whetherthese AdBDNF-induced neurons can functionally integrate, both withresident striatal neurons and nigrostriatal afferents, whether they cansurvive longer than 5-8 weeks, and whether they can survive the primarydisease process better than the cells they are intended to replace.These uncertainties notwithstanding, the adenoviral BDNF-mediatedinduction of neuronal addition to the adult brain expands the currentconception of cellular plasticity in the adult CNS, while lending a newperspective to the potential for gene therapy in the treatment ofstructural neurological condition.

Although preferred embodiments have been depicted and described indetail herein, it will be apparent to those skilled in the relevant artthat various modifications, additions, substitutions, and the like canbe made without departing from the spirit of the invention and these aretherefore considered to be within the scope of the invention as definedin the claims which follow.

1. A method of inducing addition of medium spiny neurons in post-nataland adult brain comprising: providing a neurotrophic factor selectedfrom the group consisting of NT-4 and brain-derived neurotrophic factor;providing a bone morphogenic protein inhibitor; and injecting theneurotrophic factor and the bone morphogenic protein inhibitor into asubject's lateral ventricles under conditions effective to induceaddition of medium spiny neurons in any one or all of the caudatenucleus and the putamen of the subject.
 2. The method according to claim1, wherein the neurotrophic factor is NT-4.
 3. The method according toclaim 1, wherein the neurotrophic factor is brain-derived neurotrophicfactor.
 4. The method according to claim 1, wherein the bone morphogenicprotein inhibitor is noggin.
 5. A method of inducing addition of mediumspiny neurons in a subject having Huntington's Disease comprising:providing a neurotrophic factor selected from the group consisting ofNT-4 and brain-derived neurotrophic factor; providing a bone morphogenicprotein inhibitor; and injecting the neurotrophic factor and the bonemorphogenic protein inhibitor into the subject's lateral ventriclesunder conditions effective to induce addition of medium spiny neurons inany one or all of the caudate nucleus and the putamen of the subject. 6.The method according to claim 5, wherein the neurotrophic factor isNT-4.
 7. The method according to claim 5, wherein the neurotrophicfactor is brain-derived neurotrophic factor.
 8. The method according toclaim 5, wherein the bone morphogenic protein inhibitor is noggin. 9.The method according to claim 5, wherein Huntington's disease isdelayed.
 10. A method of treating Huntington's Disease comprising:providing a neurotrophic factor selected from the group consisting ofNT-4 and brain-derived neurotrophic factor; providing a bone morphogenicprotein inhibitor; and introducing the neurotrophic factor and the bonemorphogenic protein inhibitor into any one or all of a subject's caudatenucleus and putamen under conditions effective to induce addition ofmedium spiny neurons and to treat Huntington's Disease.
 11. The methodaccording to claim 10, wherein the neurotrophic factor is NT-4.
 12. Themethod according to claim 10, wherein the neurotrophic factor isbrain-derived neurotrophic factor.
 13. The method according to claim 10,wherein the bone morphogenic protein inhibitor is noggin.
 14. The methodaccording to claim 10, wherein Huntington's Disease is delayed.